539875

Oxidative Medicine and Cellular Longevity
Modulation of Oxidative Stress:

Pharmaceutical and
Pharmacological Aspects
Guest Editors: Liudmila Korkina, Tomris Ozben, and Luciano Saso
Pharmaceutical and

Pharmaceutical and
Guest Editors: Liudmila Korkina, Tomris Ozben,

and Luciano Saso
Copyright 2016 Hindawi Publishing Corporation. All rights reserved.
This is a special issue published in Oxidative Medicine and Cellular Longevity. All articles are open access articles distributed under the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Editorial Board
Mohammad Abdollahi, Iran Daniela Giustarini, Italy Valentina Pallottini, Italy
Antonio Ayala, Spain Saeid Golbidi, Canada David Pattison, Australia
Neelam Azad, USA Tilman Grune, Germany Serafina Perrone, Italy
Peter Backx, Canada Hunjoo Ha, Republic of Korea Tiziana Persichini, Italy
Damian Bailey, UK Guido Haenen, Netherlands Vincent Pialoux, France
Consuelo Borras, Spain Nikolas Hodges, UK Chiara Poggi, Italy
Vittorio Calabrese, Italy Tim Hofer, Norway Aurel Popa-Wagner, Germany
Angel Catala, Argentina Silvana Hrelia, Italy Ada Popolo, Italy
Shao-Yu Chen, USA Maria G. Isaguliants, Sweden Jose L. Quiles, Spain
Zhao Zhong Chong, USA Vladimir Jakovljevic, Serbia Kota V. Ramana, USA
Giuseppe Cirillo, Italy Peeter Karihtala, Finland Pranela Rameshwar, USA
Massimo Collino, Italy Raouf A. Khalil, USA Sidhartha D. Ray, USA
Mark J. Crabtree, UK Kum Kum Khanna, Australia Alessandra Ricelli, Italy
Manuela Curcio, Italy Neelam Khaper, Canada Francisco J. Romero, Spain
Andreas Daiber, Germany Thomas Kietzmann, Finland Vasantha Rupasinghe, Canada
Felipe Dal Pizzol, Brazil Mike Kingsley, UK Gabriele Saretzki, UK
Francesca Danesi, Italy Ron Kohen, Israel Honglian Shi, USA
Domenico DArca, Italy W. J.H. Koopman, Netherlands Cinzia Signorini, Italy
Yolanda de Pablo, Sweden Jean-Claude Lavoie, Canada Dinender K. Singla, USA
James Duce, UK Christopher Horst Lillig, Germany Richard Siow, UK
Gregory Durand, France Paloma B. Liton, USA Shane Thomas, Australia
Javier Egea, Spain Nageswara Madamanchi, USA Rosa Tundis, Italy
Amina El Jamali, USA Kenneth Maiese, USA Giuseppe Valacchi, Italy
Ersin Fadillioglu, Turkey Tullia Maraldi, Italy Jeannette Vasquez-Vivar, USA
Qingping Feng, Canada Reiko Matsui, USA Victor M. Victor, Spain
Giuseppe Filomeni, Italy Steven McAnulty, USA Michal Wozniak, Poland
Swaran J. S. Flora, India Bruno Meloni, Australia Sho-ichi Yamagishi, Japan
Rodrigo Franco, USA Trevor A. Mori, Australia Liang-Jun Yan, USA
Jose Lus Garca-Gimenez, Spain Ryuichi Morishita, Japan Guillermo Zalba, Spain
Janusz Gebicki, Australia A. Mouithys-Mickalad, Belgium Jacek Zielonka, USA
Husam Ghanim, USA Hassan Obied, Australia
Laura Giamperi, Italy Pal Pacher, USA
Contents
Modulation of Oxidative Stress: Pharmaceutical and Pharmacological Aspects
Liudmila Korkina, Tomris Ozben, and Luciano Saso
Volume 2016, Article ID 6023417, 3 pages
Skin Antiageing and Systemic Redox Effects of Supplementation with Marine Collagen Peptides and
Plant-Derived Antioxidants: A Single-Blind Case-Control Clinical Study
Chiara De Luca, Elena V. Mikhalchik, Maxim V. Suprun, Michael Papacharalambous, Arseniy I. Truhanov,
and Liudmila G. Korkina
1,4-Dihydropyridine Derivatives: Dihydronicotinamide AnaloguesModel Compounds Targeting

Oxidative Stress
Astrida Velena, Neven Zarkovic, Koraljka Gall Troselj, Egils Bisenieks, Aivars Krauze, Janis Poikans,
and Gunars Duburs
Redox Control of Multidrug Resistance and Its Possible Modulation by Antioxidants

Aysegul Cort, Tomris Ozben, Luciano Saso, Chiara De Luca, and Liudmila Korkina
Is Modulation of Oxidative Stress an Answer? The State of the Art of Redox Therapeutic Actions in
Neurodegenerative Diseases
Valerio Chiurchiu, Antonio Orlacchio, and Mauro Maccarrone
Evidence for Detrimental Cross Interactions between Reactive Oxygen and Nitrogen Species in Lebers
Hereditary Optic Neuropathy Cells
Micol Falabella, Elena Forte, Maria Chiara Magnifico, Paolo Santini, Marzia Arese, Alessandro Giuffre,
Kristina Radic, Luciana Chessa, Giulia Coarelli, Maria Chiara Buscarinu, Rosella Mechelli,
Marco Salvetti, and Paolo Sarti
Exercise Modulates Oxidative Stress and Inflammation in Aging and Cardiovascular Diseases
Nada Sallam and Ismail Laher
Cardiovascular and Hepatic Toxicity of Cocaine: Potential Beneficial Effects of Modulators of Oxidative
Stress
Manuela Graziani, Letizia Antonilli, Anna Rita Togna, Maria Caterina Grassi, Aldo Badiani,
and Luciano Saso
Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli

Katarzyna Licznerska, Bozena Nejman-Falenczyk, Sylwia Bloch, Aleksandra Dydecka, Gracja Topka,
Tomasz Gasior, Alicja Wegrzyn, and Grzegorz Wegrzyn
The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting
Prophages
Katarzyna Licznerska, Aleksandra Dydecka, Sylwia Bloch, Gracja Topka, Bozena Nejman-Falenczyk,
Alicja Wegrzyn, and Grzegorz Wegrzyn
Modulation of Hypercholesterolemia-Induced Oxidative/Nitrative Stress in the Heart

Csaba Csonka, Marta Sarkozy, Marton Pipicz, Laszlo Dux, and Tamas Csont
Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk
Activity in Mouse Embryonic Stem Cells
Jan Kucera, Lucia Bino, Katerina Stefkova, Josef Jaros, Ondrej Vascek, Josef Vecera, Lukas Kubala,
and Jir Pachernk
Protective Effects of D-Penicillamine on Catecholamine-Induced Myocardial Injury

Michal Rha, Pavlna Haskova, Jan Martin, Tomas Filipsky, Katerina Vanova, Jaroslava Vavrova,
Magdalena Holeckova, Pavel Homola, Libor Vtek, Vladimr Palicka, Tomas Simunek,
and Premysl Mladenka
A New Method to Simultaneously Quantify the Antioxidants: Carotenes, Xanthophylls, and Vitamin A
in Human Plasma
Mariel Colman-Martnez, Miriam Martnez-Huelamo, Esther Miralles, Ramon Estruch,
and Rosa M. Lamuela-Raventos
Relation between Endothelial Nitric Oxide Synthase Genotypes and Oxidative Stress Markers in Larynx
Cancer
K. Yanar, U. Cakatay, S. Aydn, A. Verim, P. Atukeren,
N. E. Ozkan, K. Karatoprak, T. Cebe, S. Turan, E. Ozkok,
G. Korkmaz, C. Cacna, O. Kucukhuseyin, and I. Yaylm
The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

Andreia Ascenso, Tiago Pedrosa, Sonia Pinho, Francisco Pinho, Jose Miguel P. Ferreira de Oliveira,
Helena Cabral Marques, Helena Oliveira, Sandra Simoes, and Conceicao Santos
Mitochondrion-Permeable Antioxidants to Treat ROS-Burst-Mediated Acute Diseases

Zhong-Wei Zhang, Xiao-Chao Xu, Ting Liu, and Shu Yuan
Hindawi Publishing Corporation
http://dx.doi.org/10.1155/2016/6023417
Editorial
Modulation of Oxidative Stress: Pharmaceutical and
Liudmila Korkina,1 Tomris Ozben,2 and Luciano Saso3

1
Centre of Innovative Biotechnological Investigations (CIBI-Nanolab), 197 Vernadskogo Prospekt, Moscow 119571, Russia
2
Department of Biochemistry, Akdeniz University Medical Faculty Campus, Dumlupnar Street, 07070 Antalya, Turkey
3
Department of Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, Piazzale Aldo Moro 5,
00185 Rome, Italy
Correspondence should be addressed to Luciano Saso; luciano.saso@uniroma1.it
Received 11 January 2016; Accepted 12 January 2016
Copyright 2016 Liudmila Korkina et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Notwithstanding the fact that the multiple roles of oxidative The results indicated a potential relationship among G894T
stress in human biology and pathology have been intensely polymorphism of NOS3 and impaired redox homeostasis that
discussed over the last half century, the problem is still far may influence the risk of laryngeal cancer.
beyond our full comprehension. Thus, in a comparatively An interesting mechanism of adaptation to oxidative
short history of oxidative medicine, the roles of two major stress evolved in enterohemorrhagic Escherichia coli, consist-
heroes, free radicals and antioxidants, have been entirely ing of upregulation of Shiga toxin production by prophages
redefined. Free radicals and other reactive oxygen and nitro- of the bacteria in response to H2 O2 excretion by infected
gen species, widely recognized two-three decades ago as human neutrophils, was described by K. Licznerska and
absolute evils leading to and/or accompanying damage to coauthors. The intestinal haemorrhage induced by this type of
biologically important molecules and structures, have been bacteria depends mainly on Shiga toxin cytotoxicity. This way
recently transformed into positive actors, in the appreciation enterohemorrhagic Escherichia coli become tolerant to natu-
of their essential impact in the intracellular signaling on the ral redox-based antibacterial defense of the host organism.
organisms defense against biotic and abiotic stresses. Several One could assume that the modulation of hydrogen peroxide
original research papers published in this special issue have production by human neutrophils could be a promising strat-
been focused on this subject. egy against this type of bacterial virulence/toxicity. The role
The evidence for detrimental cross interaction of reactive of the exo-xis region of the bacterial genome in the induction
oxygen and nitrogen species was shown in a single clinical of Shiga toxin-converting prophages is further elucidated
case report of rare human disease (Lebers Hereditary Optic by the same group of authors (K. Licznerska and coauthors).
Neuropathy (M. Falabella et al.)). The chronic change in Unfortunately, the great hope that direct antioxidants
the NO homeostasis and enhanced superoxide availability could be the panacea resolving practically all health problems
contributed to dysfunction of peripheral blood mononuclear has vanished, due to the growing number of inconclusive or
cells derived from a patient due to a cooperative action of negative data from epidemiological and clinical studies. The
nitrosative and oxidative stresses in driving on the genetically current state of uncertainty regarding feasibility of antiox-
determined pathology. idant therapy is partly due to pitfalls of biologically rele-
Relations between endothelial nitric oxide synthase geno- vant and reliable methods of determination of free radical
types and oxidative stress markers in patients with larynx scavenging and antioxidant capacities of isolated substances
cancer were studied in an original research (K. Yanar et al.). and compositions. The paper by M. Colman-Martnez and
2 Oxidative Medicine and Cellular Longevity
coauthors suggests a simple and accurate method for simul- adverse health effects under such acute conditions are
taneous quantification of carotenes, xanthophylls, and retinol discussed.
in human plasma based on reversed phase high-performance Negative conclusions were drawn (M. Graziani and coau-
liquid chromatography coupled with diode array detector thors) on feasibility of adjuvant therapy of cardiovascular and
(HPLC-DAD). hepatic toxicity of cocaine with modulators of oxidative stress
An innovative approach to modulate and maintain nor- (N-acetylcysteine, SOD mimetics, nitroxides and nitrones,
mal redox profile in order to achieve desirable therapeutic mitochondria targeting antioxidants, and NADPH oxidase
effects has recently become a leading one in the management and xanthine oxidase inhibitors,) notwithstanding multiple
of a variety of redox-dependent pathologies with unmet as redox fingerprints in the toxicity of drug abuse. The discrep-
yet therapeutic needs. Thus, comprehensive review by V. ancy was ascribed to pitfalls in the clinical studies design, to
Chiurchiu and coauthors attempted to answer the question the use of single instead of several targeted oxidative stress
whether modulation of oxidative stress could be therapeu- modulators, and to the lack of specific, reliable, and repro-
tically feasible in the treatment of severe neurodegenerative ducible markers of oxidative stress.
diseases, such as Alzheimers and Parkinsons diseases, amy- An outstanding comprehensive review by N. Sallam and I.
otrophic lateral sclerosis, multiple sclerosis, and hereditary Laher concentrates on recent data and modern hypotheses on
spastic paraplegia. Scrupulously analyzing redox-connected how physical exercise modulates oxidative stress and inflam-
mechanisms underlying the pathogenesis and multiple redox mation in ageing and cardiovascular diseases. A necessity for
fingerprints characteristic for these diseases, authors came to strictly individualized programs of age and cardiovascular
the conclusion that antioxidants seem to limit the oxidative pathology preventive physical exercises is based on the
tissue damage through inhibition of inflammatory events individual sensitivity to the type, intensity, frequency, and
or exerting neuroprotective properties. They also emphasize duration of physical challenge. As target organs for protective
that the main problem is embodied by the necessity to redox balancing and anti-inflammatory exercises, there are
develop antioxidants that are able to cross the blood-brain adipose tissue, skeletal muscles, immune system, and cardio-
barrier. The authors strongly consider that targeted specific vascular system components. The following molecular path-
redox modulators like dimethyl fumarate rather than con- ways mediate therapeutic and/or prophylactic effects of phys-
ventional dietary antioxidants represent a novel avenue in the ical exercise: redox-sensitive transcription factors, pro- and
management of these human diseases. anti-inflammatory cytokines, antioxidant and prooxidant
Within the same line, the effects of various pharmaceu- enzymes, and repair proteins.
ticals, nutraceuticals, some novel potential pharmacological Another review (A. Cort et al.) describes in detail a
approaches, and physical exercise on hypercholesterolemia- complex redox pattern underlying huge multiple drug resis-
induced oxidative/nitrative stress and subsequent cardiac tance (MDR) problem in antitumour therapies as well as
dysfunction are discussed in the review (C. Csonka et al.). The the development and clinical use of oxidative stress mod-
authors are focused on 3 different approaches: (i) cholesterol- ulators to combat MDR, thus enhancing efficacy of anti-
lowering therapies which attenuate oxidative/nitrosative cancer protocols. In the authors opinion, redox active drugs
stress; (ii) combined cholesterol-lowering and antioxidant (pro- or antioxidants) targeting an axis consisting of drug
protocols; and (iii) inducers of endogenous antioxidants or transporters, aryl hydrocarbon receptor, phase I/II metabolic
inhibitors of prooxidant enzymes as promising drugs for enzymes, and the inducible Nrf2-linked pathway could pro-
the prevention and/or treatment of cholesterol-induced car- vide a valid and promising way to overcome a dreadful
diac dysfunction. Among pharmaceuticals, statins, Ezetim- obstacle of MDR in cancer therapies.
ibe, niacin, fibrates, and an antidiabetic drug Rosiglitazone Reflecting the general state of the art in the development
are discussed in detail. As potent nutraceuticals, antioxi- of novel biologically available antioxidants/redox modula-
dant vitamins, coenzyme Q10, flavonoids (rutin, quercetin, tors, a majority of original research papers dedicated to
naringin, etc.), green tea catechins, and resveratrol are the testing of their pharmacological properties have been
listed. Novel modulators of miRNAs leading to attenuation presented in the in vitro systems or in vivo animal experi-
of hypercholesterolemia-induced oxidative/nitrosative stress ments. The effects of the clinically used copper chelator D-
are under development and investigation. penicillamine in the catecholamine model of acute myocar-
The review article from the Chinese group (Z.-W. Zhang dial injury were tested in cardiomyoblast cell line H9c2 and
et al.) highlights the potential of mitochondrion-permeable in Wistar Han rats (M. Rha et al.). D-Penicillamine had a
antioxidants in the management of oxidative burst-mediated protective effect against catecholamine-induced injury both
acute inflammatory conditions. The authors compare and in vitro and in vivo. The classical NOX inhibitors apocynin
evaluate well-known mitochondrion-permeable antioxi- and diphenyleneiodonium impaired proliferation of culti-
dants, such as edaravone, idebenone, alpha-lipoic acid, car- vated mouse embryonic stem cells (J. Kucera et al.) that clearly
otenoids, vitamin E, and coenzyme Q10. In addition, they reflected essential role(s) of NOX-produced reactive oxygen
focus on mitochondria-targeted MitoQ and SkQ anti- species as signalling agents for embryonic cell growth and dif-
oxidants and propose astaxanthin, a carotenoid, for acute ferentiation. Preexposure of UV-B-irradiated human immor-
inflammatory conditions characterized by pathologically talised keratinocyte cell line (HaCaT) to complexed lycopene
increased oxidative burst, for example, avian influenza with (A. Ascenso et al.), a skin located antioxidant, led to a shift
acute respiratory distress syndrome and ischemia-reper- of the ratio dead : apoptotic : viable subpopulations towards
fusion. The limitations of the antioxidant use including normal values. The authors concluded that the complexed
Oxidative Medicine and Cellular Longevity 3
lycopene might have protective or cytotoxic effects in pho-

todamaged and preneoplastic keratinocytes, while allow-
ing other keratinocytes to accelerate repairing mechanisms
and remain viable. A broad group of 1,4-dihydropyridine
derivatives as long time known compounds with antioxidant
potential have been extensively evaluated during last three
decades. A. Velena and coauthors attempt to explain the
innovative interest in these agents for biomedical appli-
cations. Examples of protective and antioxidant properties
of 1,4-dihydropyridine derivatives have been provided in a
number of recent in vitro studies on low density lipoproteins,
mitochondria, microsomes, isolated cells, and cell cultures.
As expected, submissions of clinical data were scarce.
Only one paper (C. De Luca and coauthors) depicted results
of the clinical-laboratory study on the skin antiageing and
systemic redox effects of supplementation with the compo-
sition of marine collagen peptides and plant-derived skin-
targeting antioxidants (coenzyme Q10 + grape skin extract +
luteolin + selenium). The data obtained clearly show the
improvement of skin properties (elasticity, sebum produc-
tion, biological age, and dermal ultrasound markers) due
to enhanced collagen synthesis without risk of oxidative
stress that is usually connected with the synthesis. Moreover,
hormesis-like action of the supplementation allowed suggest-
ing this mechanism to be responsible for its antiageing and
energizing effects.
Due to the limited space of the special issue, we were
not able to concentrate on the issues of extreme importance
for discovery and development of safe and clinically efficient
redox modulators, such as optimization of delivery systems,
pharmacokinetics and metabolism, redox activity of metabo-
lites, and biological markers for the assessment of in vivo
prooxidant/antioxidant action correlating with clinical out-
comes.
Liudmila Korkina
Tomris Ozben
Luciano Saso
http://dx.doi.org/10.1155/2016/4389410
Clinical Study
Skin Antiageing and Systemic Redox Effects of Supplementation
with Marine Collagen Peptides and Plant-Derived
Antioxidants: A Single-Blind Case-Control Clinical Study
Chiara De Luca,1 Elena V. Mikhalchik,2 Maxim V. Suprun,2

Michael Papacharalambous,3 Arseniy I. Truhanov,4 and Liudmila G. Korkina4,5
1
Evidence-Based Well-Being Ltd., 31 Alt-Stralau, 10245 Berlin, Germany
2
Russian Institute of Physical Chemical Medicine, 1A Malaya Pirogovskaya Street, Moscow 117513, Russia
3
Dermatology Clinic Orthobiotiki Clinical, 3-5 Sorou Street, 15125 Athens, Greece
4
Active Longevity Clinic, Beauty Institute on Arbat, 8 Maliy Nikolopeskovskiy Lane, Moscow 119002, Russia
5
Centre of Innovative Biotechnological Investigations (CIBI-Nanolab), 197 Vernadskogo Prospekt, Moscow 119571, Russia
Correspondence should be addressed to Liudmila G. Korkina; korkina@cibi-nanolab.com
Received 5 August 2015; Revised 6 December 2015; Accepted 24 December 2015
Academic Editor: Tiziana Persichini
Copyright 2016 Chiara De Luca et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Recently, development and research of nutraceuticals based on marine collagen peptides (MCPs) have been growing due to their
high homology with human collagens, safety, bioavailability through gut, and numerous bioactivities. The major concern regarding
safety of MCPs intake relates to increased risk of oxidative stress connected with collagen synthesis (likewise in fibrosis) and to ROS
production by MCPs-stimulated phagocytes. In this clinical-laboratory study, fish skin MCPs combined with plant-derived skin-
targeting antioxidants (AO) (coenzyme Q10 + grape-skin extract + luteolin + selenium) were administered to volunteers ( = 41).
Skin properties (moisture, elasticity, sebum production, and biological age) and ultrasonic markers (epidermal/dermal thickness
and acoustic density) were measured thrice (2 months before treatment and before and after cessation of 2-month oral intake). The
supplementation remarkably improved skin elasticity, sebum production, and dermal ultrasonic markers. Metabolic data showed
significant increase of plasma hydroxyproline and ATP storage in erythrocytes. Redox parameters, GSH/coenzyme Q10 content,
and GPx/GST activities were unchanged, while NO and MDA were moderately increased within, however, normal range of values.
Conclusions. A combination of MCPs with skin-targeting AOs could be effective and safe supplement to improve skin properties
without risk of oxidative damage.
1. Introduction dietary substances with potential antiageing efficacy is their

low bioavailability due to limited and selective penetration
Dietary vitamins, plant-derived polyphenols, fatty acids, pro- through the intestinal barrier, destruction by the intestinal
teins, essential amino acids, and trace elements have demon- microorganisms, high rate of metabolism, and preferential
strated beneficial effects on skin health and appearance [1 distribution between tissues and organs [6, 7].
3]; hence, the use of nutraceuticals targeting skin is stead- Marine fish collagen and collagen peptides have been
ily growing. For example, photoprotection by intake of dieta- widely used as functional foods or dietary supplements due to
ry antioxidants has been a subject of numerous in vitro, their homology to human collagen structure [8], safety profile
animal, and human studies [4, 5]. Within this direction, the [9], stability, biocompatibility, high bioavailability through
search for reliable and effective antiageing remedies for gastrointestinal barrier [10], and potent bioactivities [11].
both topical and systemic administration has become a hot Marine collagen peptides (MCPs) obtained by enzymatic
spot for cosmetic, food, and biomedical companies. One digestion of fish skin have been shown to exert several health
of the strongest limitations for skin-targeting plant-derived effects mainly in two directions: metabolic disorders and
skin/bone repair. Thus, they positively affected glucose and positive roles of mediators in the MCPs-induced collagen
lipid metabolism in patients with type II diabetes mellitus and ATP synthesis/storage, as well as in sebum production.
[12], improved lipid metabolism in obese people [13] and ge- On these grounds, we suggested that selected antioxidants
netically modified mice [14], ameliorated early alcoholic liver targeting the distinct organs/tissues should be essential
injury [15], and possessed hypotensive and lipid normalising components of MCPs-containing nutraceuticals for more
action in patients with primary hypertension [16]. A great effective, individualised, and safe supplementation.
majority of publications demonstrated significant wound
healing efficacy of orally administered MCPs in animal 2. Materials and Methods
models of excision and full-thickness skin wounds [10, 17, 18].
Recently, collagen peptides isolated by enzymatic digestion 2.1. Patients. The study enrolled a group of 41 adult healthy
from fish, bovine, and porcine skin as well as from chicken Caucasian volunteers of both sexes recruited from the Beauty
and bovine cartilage have drawn particular interest for the Institute on Arbat (Moscow, Russia) staff (age 3772 years;
treatment of patients with osteoarthritis. Several clinical trials mean age 50.6 10.4 years; 5 males and 36 females) following
showed that MCPs were safe and provided an improvement the exclusion and inclusion criteria for an open single-blind
in terms of pain and functions in such patients [19]. From clinical study. The inclusion criteria were as follows: (i)
mechanistic point of view, the oral intake of MCPs stim- healthy white adult subjects of both sexes, 3575 years of age,
ulated the synthesis of extracellular matrix (ECM) macro- (ii) subjects with visible symptoms of aged facial skin, (iii)
molecules such as endogenous collagen, by upregulating gene subjects who agreed to interrupt any intake of antioxidant
expression of several collagen-modifying enzymes involved nutraceuticals/drugs for at least 1 week before and during
in posttranslational collagen modification and cross-linking the entire duration of the trial, and (iv) subjects without any
[20]. Several in vitro studies have shown antioxidant prop- difficulty to understand and follow the clinical investigator
erties of very-low-molecular-weight (120 Da) MCPs [21, 22] instructions. Pregnant and breastfeeding women, subjects
containing proline, which is a scavenger of hydroxyl radicals. with allergic/intolerance reactions to any component of the
Of importance for the present study, MCPs are considered tested product, subjects on any other nutraceutical interven-
antiageing compounds because they seem to increase life tions or/and therapies, and subjects simultaneously engaged
span in rats by inhibiting spontaneous tumour incidence [9], in other clinical trials were excluded from the study. The
possess photoprotective and immunomodulating properties participants were informed that they could interrupt clinical
[2325], and improve/eliminate signs of premature senes- trial at any moment, without any explanation of causative
cence of human skin [26, 27]. reason for their action, or in case they noticed any adverse
The major concern regarding safety and clinical feasibility reaction to the tested product or had any sensation that the
of regular intake of MCPs has been raised from the well estab- product intake affected their appearance negatively.
lished fact that the induction of collagen synthesis, mainly The protocol of the clinical trial was duly analysed and
assessed by the increased hydroxyproline levels, is often approved by the Ethical Committee of the Beauty Insti-
associated with oxidative stress [2830]. Moreover, MCPs of tute on Arbat, Moscow, Russia (number 11/EK-2014). All
different origin have been shown to activate innate immune recruited subjects gave their informed consent to personal
response of macrophages and neutrophils through Toll-like and anamnestic data collection and biological material sam-
receptor 4, which leads to NADPH-oxidase (NOX4) activa- pling. The guidelines of Helsinki Declaration for human
tion and reactive oxygen species overproduction [31, 32]. A experimentation were strictly followed during the conduct of
newly developed composition of MCPs with a complex of the clinical trial.
essential skin-targeting antioxidants, that is, coenzyme Q10 +
selenium + luteolin + grape-skin extract, demonstrated UVA- 2.2. Food Supplement under Investigation. Food supplement
protective effects in the preliminary in vitro experiments on containing marine collagen peptides derived from skin of
human skin biopsies [25]. However, the composition under deep sea fish (MCPs, 570 mg), grape-skin extract (10 mg),
commercial name of CELERGEN has never been evaluated coenzyme Q10 of plant origin (10 mg), luteolin (10 mg), and
clinically when administered as a food supplement. selenium (0.05 mg) of plant origin was formulated in soft
The goal of the present clinical-laboratory study was to gelatine capsules. As inactive solvents, refined and partly
elucidate the effects of the oral administration of CELERGEN hydrogenated soybean oil as well as small admixture of pure
on skin physiology and dermal collagen deposition in the soybean lecithin were used. The product, under the commer-
group of healthy middle-aged subjects with clinical signs cial name of CELERGEN (manufacturer: Laboratories-Dom,
of skin ageing. The cutaneous clinical-instrumental data Carouge, Switzerland), was kindly provided by Suisse Ueli
were compared with the systemic metabolic parameters of Corporation. According to the manufacturers information,
collagen synthesis, redox balance, and energy storage. For the the deep sea fish sources, that is, Pollachius virens, Hippoglos-
first time, we demonstrated (i) remarkable improvement of sus hippoglossus, and Pleuronectes platessa, originated from
ageing skin physiology and structure, which corresponded the French coast of the North Sea.
to enhanced systemic markers of collagen synthesis; (ii) Fish skin was homogenised in distilled water, with
systemic redox balance, sustained by the antioxidant com- addition of complex proteases. The enzymatic proteolytic
plex; and (iii) increased systemic energy storage. We also process was carried out at 40 C and pH 8.0 for 3 h, after
hypothesised that moderately increased plasmatic levels of which the proteases were inactivated by short-term heating
nitric oxide (NO) and malonyl dialdehyde (MDA) may play (56 C for 10 min). The liquid was sterilised by Millipore
filtration (pore size 0.02 mm) and spray-dried to prepare PLUS technique. The transepidermal water loss (TEWL), an
MCP powder, as described in detail previously [17, 33]. index of skin moisture, was assessed with Tewameter, which
Chemical analysis by Kjeldahl assay of the powder confirmed measures the water evaporation through cutaneous levels.
a >90% content of collagen peptides, with moisture and ash When the skin is aged or damaged, the barrier properties of
content <10%. According to previous publications [10, 33, the skin are affected, with increased water evaporation and
34], the molecular weight distribution of MCPs after the reduced skin hydration. Sebum content was measured by the
described enzymatic digestion process was within the range SOFT PLUS sebometric probe.
of 1060 Da, and MCPs were enriched in glycine, glutamine,
proline, hydroxyproline, asparagine, alanine, and arginine. 2.5. Assessment of Ultrasound Properties of the Skin. Assess-
The aqueous extract of grape skin was obtained from Vitis ment of ultrasound properties of the skin was performed by
vinifera Linn. fruit and contained at least 70% of polyphenols a digital ultrasound imaging system DUB CUTIS (Digital
and 20% of procyanidins as per UV/Vis spectrophotometry Ultraschall Bildsystem, Germany), which allowed determin-
data. The coenzyme Q10 component of plant origin was ing four parameters simultaneously: epidermal and dermal
of highest purity (100 3%), confirmed by both IR spec- thickness and epidermal and dermal ultrasonic density. The
trophotometry and high performance liquid chromatography first two parameters are indirect markers of collagen (dermis)
(HPLC) methods. Food-quality luteolin was extracted from and lipid (epidermis) synthesis and retention while the
Marigold plant petals, and the extract contained 20% of second pair of parameters characterises the evenness and
luteolin and 1% of zeaxanthin evaluated by HPLC analysis. order in the epidermal and dermal structures, respectively.
Selenium in the form of selenite (according to gravimetric The elastic properties of the skin were additionally analysed
method) was extracted from plant bulbs and leaves. Acute by a TPM system containing elastometric sensor (22 MHz)
and chronic toxicity data and documents of Certificates of which combines digital ultrasound examination with an
Analyses, Security, and Registration in Switzerland were duly imaging record (DUB CUTIS, Germany). A computerised
provided by the manufacturer. multifunctional diagnostic tool integrating different morpho-
metric parameters (epidermal thickness, tone, wrinkles, and
2.3. Clinical Study Design. The entire trial duration was 4 elasticity) for face skin biological age determination was used
months (MayDecember 2014), that is, 2 months of pretreat- (SOFT PLUS TOP, Callegari, Parma, Italy).
ment period, followed by 2 months of treatment with the
test nutraceutical administration. The facial skin parameters 2.6. Reagents and Assay Kits. The majority of chemical
of recruited volunteers were analysed three times: at the reagents, HPLC standards, mediums, solvents, and luciferin-
first visit (enrollment), at the second visit 2 months after luciferase for ATP assay were from Sigma Chemical Co. (St.
the pretreatment period, and at the third visit immediately Louis, MO, USA); kits for enzyme activity assays and Griess
after the treatment period. Each assessment session com- reagent for nitrites/nitrates determination were from Cay-
prised instrumental methods for measuring skin physiology man Chemical Company (Ann Arbor, MI, USA). Manufac-
parameters and ultrasound properties of the skin layers. This turers of other reagents are mentioned within the respective
design allowed us to use the same subject as a control and methods.
an experiment. During the treatment period, the volunteers
were recommended to take 2 capsules of CELERGEN a
2.7. Redox and Oxidation Markers Studies. Complete differ-
day (at breakfast and dinner time) for 60 consecutive days.
ential blood cell counts and metabolic analyses were per-
At the second and third visits, the participants donated
formed on fresh ethylenediaminetetraacetic acid- (EDTA-)
20 mL of venous blood after overnight fasting and test tubes
anticoagulated venous blood of 12 hrs fasting subjects. Bio-
were coded by the principal clinical investigator. Blood
chemical assays were performed on peripheral blood plasma
samples were routinely processed for general haematology
or red blood cells (RBC), either immediately (ATP, glu-
(haemoglobin content, differential cell count, and the rate of
tathione, and coenzyme Q10 ) or within 72 hrs, on sample
erythrocyte sedimentation) and biochemistry (glucose levels,
aliquots stored at 80 C under argon. Plasma levels of
plasma protein and lipid profiles, transaminases activities,
nitrites/nitrates (NO2 /NO3 , expressed as moles/L) were
and C-reactive protein content). Laboratory operators carried
measured spectrophotometrically by Griess reagent [35]. Pro-
out analytical determinations blindly, and statistician was not
tein content was measured according to Bradford [36], using
informed which set of analyses was done in the control or
a microplate assay kit (Bio-Rad, Hercules, CA, USA). Total
experimental periods, hence ranking this study of clinical
glutathione (reduced + oxidized glutathione, GSH + GSSG,
efficacy of the nutraceutical as a single-blind clinical inves-
mg/g Hb) levels in erythrocytes were measured by HPLC
tigation.
(Shimadzu Scientific Instruments, Columbia, MD, USA)
according to Reed et al. [37]. Total coenzyme Q10 (CoQ10 H2 +
2.4. Assessment of Skin Physiology Parameters. Several phys- CoQ10 , mg/L) levels in plasma were quantified by HPLC
iological parameters, mainly barrier properties, of the facial as described previously [38]. In brief, 1 mL plasma sample,
skin were assessed by appropriate SOFT PLUS TOP probes, with adequate amount of coenzyme Q9 (internal standard)
with microcamera visual analysis and patented comput- and 500 L acetic acid (50% solution), was extracted twice,
erised programs (Callegari, Parma, Italy). Skin elasticity was first with 3.5 mL and then with 2.5 mL of ethanol/hexane
determined by the elastometric approach used in the SOFT mixture (2 : 5 vol/vol), with homogenisation and subsequent
Table 1: Subjective evaluation of the 2-month food supplement administration effects, by participants ( = 41).
Number (%) of participants

Parameter
Improvement No effect Aggravation
General health conditions 21 (51%) 20 (49%) 0 (0%)
Stamina/muscle strength/joint motility 15 (36%) 26 (64%) 0 (0%)
Digestive system 0 (0%) 41 (100%) 0 (0%)
Skin conditions 25 (61%) 16 (39%) 0 (0%)
centrifugation. The upper phase containing hexane extract counter, equipped with Wallac 1420 Software (Perkin Elmer,
was evaporated under nitrogen flux and then resuspended in MA, USA). Results were expressed as mmoles/L.
an adjusted amount of a methanol/isopropanol (3 : 2 vol/vol)
mixture for HPLC analysis. Reduced and oxidized forms of 2.9. Hydroxyproline Assay. The plasma levels of free hydrox-
coenzyme Q10 (CoQ10 H2 and CoQ10 ) were quantified simul- yproline (Hyp) and hydroxyproline in the form of oligopep-
taneously with HPLC equipped with analytical Supelcosil LP- tides, mainly proline-hydroxyproline, were determined by
18 column (24 cm 4.6 mm, 5 m, Supelco, Bellefonte, PA, a chemical colorimetric method using a commercial kit
USA) plus its guard column, and in line photodiode array (Hydroxyproline Detection Kit) in accord with the manufac-
and electrochemical detector (ESA CoulArray, Bedford, MA, turers instructions. Hyp concentrations were quantified in
USA) in accord with previously published methods [39, 40]. the linear range of its calibration curve using an array reader
The clinical normality range was extrapolated from the above (Bio-Rad, Hercules, CA, USA) and expressed in g/mL of
publications. plasma.
Plasmatic Cu,Zn-superoxide dismutase 3 (Cu,Zn-SOD3,
U/g protein) activity was measured spectrophotometrically 2.10. Statistical Analysis. Statistical analysis of clinical data
at 505 nm using appropriate kit from Cayman Chemical was carried out using WINSTAT programs for personal
Company (Ann Arbor, MI, USA) [41, 42]. RBC were lysed computers (Statistics for Windows 2007, Microsoft, USA).
in hypotonic solution and the postspin cell lysates were anal- All biochemical and molecular measurements were done in
ysed. Total RBC glutathione-S-transferase (GST, U/mg Hb) triplicate and data were statistically evaluated. Values were
activity was measured spectrophotometrically by the meth- presented as mean, standard error of the mean, and 1.96
ods described previously, using chloro-2,3-dinitrobenzene standard error of triplicate analyses. When several datasets
as substrate [43]. RBC glutathione peroxidase (GPx, U/g were compared, data were analysed by Students -test for
Hb) activity was determined using Cayman Chemical kit, unpaired data. Differences between initial/final data for a
according to the method [44]. single participant were analysed by paired -test and by
Plasma levels of MDA were determined by slightly Mann-Whitney test for changes from baseline. All reported
modified spectrophotometric analysis of thiobarbituric acid- values are from two-tailed tests, and values of less than
reactive substances (TBARS) described elsewhere [45]. After 0.05 were considered to indicate statistical significance.
a 15 min treatment of plasma (200 L) with trichloroacetic
(1.22 M) and hydrochloric (0.6 M) acids, alkaline solution
of TBA was added and the mixture was boiled for 30 min. 3. Results
TBARS were extracted with butanol and analysed spec- 3.1. Subjective Evaluation by Participants and Clinical Inves-
trophotometrically at 535 nm. The results were expressed in tigators. All healthy volunteers ( = 41) recruited in the
M of MDA using the appropriate calibration curve. trial duly completed it. There were no drop-offs due to
low compliance or adverse effects of the supplementation.
2.8. ATP Measurement in Erythrocytes. 100 L of erythrocyte Routine haematological and biochemical analyses, which
pellet was stored on ice until analysis. Ice-cold water (990 L) were carried out after blood donation in the beginning and
was added to 10 L of the erythrocytes pellet and mixed after the cessation of the study, did not show statistically
and the lysed erythrocytes were kept on ice. The principle significant changes possibly reflecting adverse consequences
of ATP assay is based on the quantitative bioluminescent of the test nutraceutical in the prescribed dosages (data not
determination of adenosine 5 -12 triphosphate (ATP), assessed shown). The subjective evaluation of the product effects on
by the Bioluminescence Assay Kit. In the assay, ATP is selected general health parameters is shown in Table 1. The
consumed when firefly luciferase catalyses the oxidation of participants were predominantly satisfied with the effects
D-luciferin to adenyl-luciferin which, in the presence of obtained on general health conditions and skin properties
oxygen, is converted to oxyluciferin with light emission. This and partly also by enhanced muscle strength and stamina. No
second reaction is essentially irreversible. When ATP is the effect whatsoever on digestion was registered.
limiting reagent, the light emitted is proportional to the ATP
present. The measurements of luciferin-luciferase chemilu- 3.2. Effects on Facial Skin Properties. Comparison of digital
minescence were performed on a Victor2 1420 multilabel photos taken before and after clinical trial showed visible
(a) (b)
(c)
Figure 1: Digital images of facial skin ultrasound examinations (patient number 23, e.g.), made 2 months before the beginning of the trial
(a), at the day of the trial beginning (b), and immediately after the trial cessation (c).
qualitative improvement of aesthetic aspect of face with Table 2: Effects of the 2-month food supplement administration on
pronounced lifting effect (data not shown). the ultrasonic properties of the dermis ( = 41).
Characteristic digital images of ultrasound examinations
Dermis
made at trial beginning (2 months before the beginning of
Parameter Pretreatment Before After
supplementation), at the first day of nutraceutical adminis-
period treatment treatment
tration, and immediately after the trial cessation are shown,
respectively, in Figures 1(a), 1(b), and 1(c). The individual Thickness, m 3884 30 3900 31 4133 28
ultrasonic characteristics were rather stable and were not Acoustic density 5.2 0.2 5.1 0.2 6.3 0.1

subjected to statistically significant changes during the 2 < 0.05 versus before treatment.
months of pretreatment period (Tables 2 and 3; compare
columns 1 and 2). The analysis of individual data showed
Table 3: Effects of the 2-month food supplement administration on
that highly enhanced dermal thickness and homogenous
the ultrasonic properties of the epidermis ( = 41).
distribution of collagen fibers in dermis were detectable in
23% ( = 11) of the participants after the trial cessa- Epidermis
tion. Statistical evaluation of dermal thickness and acoustic Parameter Pretreatment Before After
density revealed significant changes exclusively at the third period treatment treatment
visit (Table 2), while the ultrasonic properties of epidermis Thickness, m 76.9 1.0 77.0 0.8 77.6 0.9
remained unchanged (Table 3).
Acoustic density 35.6 2.4 35.2 2.2 35.4 2.0
Analyses of the main physiological parameters of the
skin relevant to ageing, such as elasticity, moisture, and
sebum content, demonstrated their comparative stability in
the pretreatment period, as there were no significant changes age, calculated on the basis of ultrasound and cutaneous
between the first and the second sets of measurements physiology measurements, tended to decrease after the trial;
(Table 4, columns 1 and 2). Conversely, CELERGEN adminis- however, the difference did not reach statistical significance.
tration statistically significantly enhanced skin elasticity and It should be noticed that all tested parameters of skin
sebum production ( < 0.0001), whilst not influencing physiology and structure were not subjected to temporal
cutaneous moisture (Table 4, columns 2 and 3). Biological fluctuations during the 2-month pretreatment period, and
Table 4: Effects of the 2-month food supplement administration on the parameters of skin physiology ( = 41).
Parameter Pretreatment period Before treatment After treatment

Elasticity 34.06 1.54 33.66 1.21 40.26 0.87
Moisture 48.83 3.02 49.03 3.52 46.54 3.02
Sebum 29.89 4.16 29.37 4.76 56.86 4.04
Skin biological age 50.11 1.91 49.51 1.68 48.09 1.74
< 0.0001 versus before treatment.
therefore changes observed can be viewed as a result of are essential for collagen biosynthesis, maturation, mode
CELERGEN administration. of deposition, and collagen fiber structure. Dietary proline
intake promotes tissue repair in humans and animals [48].
3.3. Plasmatic Oxidation Markers and Antioxidants. Sur- Recently, Wang et al. [17] reported the experimental evidence
prisingly, CELERGEN administration did not affect several that MCPs might improve collagen synthesis and maturation
markers of glutathione metabolism such as total glutathione by inducing the expression of transforming growth factor
levels (normality range: 0.51.6 mg/g Hb) and glutathione-S- beta-1 (TGF-1) and basic fibroblast growth factor (bFGF).
transferase and glutathione peroxidase activities (normality Our data (Figure 4(a)) are consistent with previously pub-
ranges: 0.20.7 U/mg Hb and 18.054.0 U/g Hb, resp.) (Fig- lished ones on rats fed with MCPs from salmon or trout
ures 2(a), 2(b), and 2(c)). At the same time, nitrite/nitrate and skin [49], showing that plasma levels of free and dipeptide
MDA levels in plasma (normality ranges: 70.3221.0 M and (Pro-Hyp) forms of hydroxyproline were highly increased
1.02.2 M, resp.) were statistically significantly increased after single intake of MCPs in soybean oil. Similar data on
( < 0.05 and < 0.0001, resp.), although they remained the blood levels of Hyp and Hyp-containing peptides were
within normal physiological range established in our lab- obtained on healthy human volunteers [50].
oratory (Figures 3(a) and 3(b)). Extracellular Cu,Zn-SOD3 Numerous animal studies on the effects of oral adminis-
activity was slightly suppressed ( < 0.001) but did not drop tration of natural or synthetic antioxidants towards collagen
below the normality border (5.020.1 U/mL) (Figure 3(c)). deposition, reactive species levels, and antioxidant defences
generated highly conflicting data, depending on the exper-
3.4. Parameters of Collagen and ATP Metabolism. Plasma imental system. Thus, with various wound healing models,
content of hydroxyproline was found highly elevated ( < it was repeatedly demonstrated that either complex plant
0.01) (Figure 4(a)), the same with ATP content in erythro- extracts containing active secondary metabolites (triterpenes,
cytes ( < 0.001) (Figure 4(c)), although total content of polyphenols, alkaloids, etc.) [18, 51] or a composition of
coenzyme Q10 was not changed after supplementation with collagen inducing polysaccharides like chitosan and antiox-
the coenzyme Q10 -containing nutraceutical (Figure 4(b)). idants such as curcumin [52] or resveratrol [53] ameliorated
wound healing increasing skin collagen deposition, while
4. Discussion suppressing proinflammatory iNOS and myeloperoxidase,
decreasing pathologically elevated levels of MDA and hydro-
In a preliminary ex vivo study of CELERGEN components gen peroxide, and improving enzymatic antioxidant defence.
against UVA-induced damage in human skin biopsies and Recent studies showed that collagen peptides from fish skin
fibroblasts [25], marine collagen peptides but not the complex remarkably promoted both wound healing and angiogenesis
of plant-derived antioxidants inhibited transcriptional and in different experimental settings [10, 17]. Of importance,
posttranscriptional matrix metalloproteinase-1 and elastase excessive NO produced during the inflammatory phase
upregulation, leading the authors to hypothesise clinical of wound healing process impaired collagen accumulation
feasibility for the prevention of skin photoaging. In contrast, [54], while moderate NO levels accelerated the granulation
another publication demonstrated that the bioflavonoid lute- phase of wound closure [18, 55]. Moreover, wound healing
olin, a component of the CELERGEN antioxidant complex, acceleration by moderate levels of H2 O2 through induction
effectively attenuated UVB-induced DNA damage, inflam- of vascular endothelial growth factor in keratinocytes and
mation, and ROS overproduction in skin cells in vitro and in macrophages was proved in a number of experimental and
vivo [46]. clinical studies [56, 57]. Here, we found that, along with Hyp
In the present study, we obtained convincing clinical accumulation, plasma levels of nitrites and nitrates, related
data on the efficacy of the marine collagen peptide and to NO production in the bloodstream, were moderately
plant antioxidant formulation CELERGEN in improving increased after CELERGEN treatment, though remaining
dermal collagen deposition and structure (Table 2), as well within the range of normal values (Figure 3(a)). Similar
as skin elasticity (Table 4). These effects were consistent results were obtained with plasmatic MDA (Figure 3(b)). This
with enhanced plasma levels of hydroxyproline, a systemic allowed us to suggest that redox regulation of cutaneous
metabolic marker of collagen synthesis (Figure 4(a)). Nearly collagen synthesis process or/and fibroblast proliferation acti-
100% of human Hyp is in fact found in collagen [47]. Hyp vation could have occurred due to physiologically relevant
being an oxidative derivative of proline, both amino acids NO and/or MDA amounts generated following supplement
Glutathione (GSH + GSSG, RBC, mg/g Hb) GST activity (RBC, U/mg Hb)
1.20 0.48
1.16 0.46
1.12 0.44
1.08 0.42
1.04 0.40
1.00 0.38
0.96 0.36
Before After Before After
Mean Mean
Mean ES Mean ES
Mean 1.96 ES Mean 1.96 ES
(a) (b)
GPx activity (RBC, U/g Hb)
70
60
50
40
30
20
10
Before After
Mean
Mean ES
Mean 1.96 ES
(c)
Figure 2: Glutathione cycle parameters: erythrocyte levels of total glutathione (reduced and oxidized forms, GSH + GSSG) (a) and erythrocyte
enzymatic activities of glutathione-S-transferase (b) and of glutathione peroxidase (c) in the study group of patients ( = 41), before and after
food supplement administration period. Values are represented as mean (), standard error of the mean (upper and lower limits of the box),
and 1.96 standard error (upper and lower whiskers). GSH: reduced glutathione; GSSG: oxidized glutathione; RBC: red blood cells; Hb:
haemoglobin; GST: glutathione S-transferase; GPx: glutathione peroxidase. Reference normality range: RBC total glutathione (0.51.6 mg/g
Hb); RBC GST activity (0.20.7 U/mg Hb); RBC GPx activity (18.054.0 U/g Hb).
intake. However, the suggestion deserves further mechanistic obtained in other tissue models of fibrosis [28, 6062],
in vitro and clinical research. including skin fibrosis [29]. It seems that complex mixtures
On the other hand, in the models of cardiac fibrosis [58, of fruit extracts contained both collagen synthesis affecting
59], the significant decrease of the model-related oxidative agents and antioxidants.
stress obtained by the use of Momordica charantia fruit UV irradiation could cause skin photodamage causing
extract [58] or Fructose Chorpondiatis total flavonoids was the symptoms of premature photoageing. Evaluating the
indeed associated with simultaneous attenuation of collagen photoprotective effects of dietary MCPs isolated from jel-
deposition, as assessed by Hyp levels. Similar results were lyfish umbrella [24] or from fish scale [63] in the model
Nitrites/nitrates (NO2 + NO3 , plasma, M) MDA (plasma, M)

195 2.2
p < 0.02706
180 2.0
1.8
165
p < 0.000000001
1.6
150
1.4
135
1.2
120
1.0
105 0.8
Mean Mean
Mean ES Mean ES
(a) (b)
Cu,Zn-SOD3 (plasma, U/mL)
13
12
11
10
7
p < 0.00035
6
Before After
Mean
Mean ES
Mean 1.96 ES
(c)
Figure 3: Systemic oxidative stress markers: plasma levels of nitrites/nitrates (NO2 + NO3 ) (a), of malonyl dialdehyde (MDA) (b), and of
Cu,Zn-superoxide dismutase 3 (Cu,Zn-SOD3) (c) in the study group of patients ( = 41), before and after food supplement administration
period. Values are represented as mean (), standard error of the mean (upper and lower limits of the box), and 1.96 standard error
(upper and lower whiskers). Intergroup significant differences () are indicated in the relative panels. NO2 + NO3 : nitrites + nitrates; MDA:
malonyl dialdehyde. Reference normality range: plasma NO2 + NO3 (70.3221.0 M); plasma MDA (1.02.2 M); plasma Cu,Zn-SOD3 (5.0
20.1 U/mL).
of chronic UVA + UVB irradiation of mice, the authors this connection, we found a significant reduction (within
concluded that MCPs enhanced skin immunity, reduced the range of normality) of plasmatic SOD3 activity follow-
water loss, restored cutaneous collagen and elastin levels and ing CELERGEN supplementation (Figure 3(c)). Extracellu-
structure, and maintained type III to I collagen ratio. Under lar plasmatic Cu,Zn-SOD3, a glycoprotein with a heparin-
similar experimental design, Zhuang et al. [64] showed the binding domain, is predominantly expressed in tissue ECM,
protective action of MCPs on antioxidant enzymes activities where it is bound to heparin sulfate proteoglycan [65].
and glutathione, lipid, and Hyp contents of murine skin. In Physiologically, SOD3 maintains redox balance and tissue
Hydroxyproline (plasma, g/mL) Coenzyme Q10 (CoQ10H2 + CoQ10 , plasma, mg/L)

1.4 0.68
p < 0.0068
1.3 0.64
1.2 0.60
1.1 0.56
1.0 0.52
0.9 0.48
0.8 0.44
Mean Mean
Mean ES Mean ES
(a) (b)
ATP (RBC, mM)
3.2
3.0 p < 0.00125
2.8
2.6
2.4
2.2
2.0
1.8
Before After
Mean
Mean ES
Mean 1.96 ES
(c)
Figure 4: Metabolic parameters related to collagen and ATP synthesis: levels of plasma hydroxyproline (a) and of the lipophilic antioxidant
total coenzyme Q10 (reduced and oxidized forms, CoQ10 H2 + CoQ10 ) (b) and erythrocyte ATP (c) in the study group of patients ( = 41),
before and after food supplement administration period. Values are represented as mean (), standard error of the mean (upper and lower
limits of the box), and 1.96 standard error (upper and lower whiskers). Intergroup significant differences () are indicated in the relative
panels. CoQ10 H2 : reduced coenzyme Q10 ; CoQ10 : oxidized coenzyme Q10 ; ATP: adenosine triphosphate; RBC: red blood cells. Reference
normality range: total coenzyme Q10 (0.41.6 mg/L); ATP (1.04.0 mM).
homeostasis and modulates innate and adaptive immune for drugs/therapeutic strategies to modulate oxidative stress
responses. Cutaneous homeostasis strongly depends on the has been drastically redirected nowadays towards (1) indirect
ECM microenvironment; therefore, an elevated SOD3 activ- AOs inducing endogenous enzymatic system of antiox-
ity may be a marker of adaptive response against intrinsic age- idative defence, mainly, through Nrf2-connected pathway;
associated and external hazardous factors inducing immune (2) selective inhibitors of ROS/RNS-producing enzymes,
suppression in the skin [66]. for example, different isoforms of NADPH-oxidase, having
Since the supplementation of compounds with a direct shown definite clinical effects; (3) recognising essential and
antioxidant effect has failed so far to show clinical efficacy and multiple physiological roles of redox balancing agents rather
sometimes even aggravated clinical picture [67], the search than mere inhibitors of free radical processes. Plant-derived
GI Circulation Skin
AOs OH
x
p-o
Hy MCPs Hyp
p Pro-Hyp Pro-Hyp
Hy
Collagen deposition
TLR4 TLR4 Elasticity
MCPs E
G
AOs M ROS, Sq-ox, PUFA-ox,
MDA, 4-HNE, and so forth
RNS NO
ROS
?
GSH
GPx AOs Sebum production
GST
SOD3
Figure 5: Scheme of the hypothesised redox-dependent mechanisms of CELERGEN physiological effects. Marine collagen peptides (MCPs)
easily penetrate gastrointestinal wall (GI, three arrows) and through blood circulation are mainly deposited in the skin. Antioxidant
component of the nutraceutical is partly metabolised in GI thus possessing low bioavailability (one arrow); however, skin-targeting
antioxidants and their metabolites reach different skin layers. While in the circulation, MCPs stimulate blood phagocytes (granulocytes
and monocytes) and endotheliocytes (E) to produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) by activating
Toll-like receptors 4 (TLR4). Hydroxyproline (HYP) and prolyl-hydroxyproline (Pro-HYP) dipeptides as major components of MCPs are
metabolised by corresponding oxidases and hydroxyl radicals are formed as by-products. Antioxidants prevent systemic oxidative stress
blocking GSH oxidation, GPx, GST, and SOD3 activation. In the skin, collagen synthesis and deposition as well as elasticity are increased while
(hypothetically) low levels of oxidised forms of skin lipids such as unsaturated fatty acids (PUFA-ox), squalene (Sq-ox), malonyl dialdehyde
(MDA), and 4-hydroxy-2-nonenal (4-HNE) may facilitate cell signalling for ATP synthesis and sebum production.
polyphenols, quercetin, resveratrol, luteolin, and many others the process of mitochondrial ATP production (Figure 4(c)).
appear to possess all these multipotent capabilities [6]. The Only excessive amounts are damaging as they initiate cell
presence of quercetin and resveratrol from grape-skin extract senescence and death. It seems that, notwithstanding coen-
and of luteolin in the antioxidant combination of CELERGEN zyme Q10 supplementation during CELERGEN course, its
may then well account for the observed redox balancing plasmatic levels were not increased (Figure 4(b)), due to
effects during the upregulation of MCPs-induced collagen elevated consumption of the coenzyme in the mitochon-
synthesis (Figures 2(a), 2(b), and 2(c)). The majority of drial cycle of energy production enhancing ATP storage in
publications have in fact demonstrated a drop of GSH content erythrocytes (Figure 4(c)) and in cell redox balance control
and an increase of protective GPx activity when Hyp content (Figures 2(a)2(c)).
was raised [28, 64, 68]. Of great importance, the presence The antiageing effects of CELERGEN supplementation
of antioxidants in the tested formulation, whilst possibly were evidenced also by the highly increased sebum pro-
protecting the redox balance from harmful side effects of duction (Table 4). It is established that the production of
collagen metabolism, did not negatively affect the desired sebaceous lipids is strongly age dependent, being low in
process of dermal collagen synthesis/deposition. In fact, the the prepubertal period, rising with sexual maturation, and
observed elevation of plasmatic Hyp was comparable with gradually declining in the aging populations (starting from
that found previously [27], with pure fish MCPs in much 4655 years) [72] or in UV-induced premature skin age-
higher dosages. ing [70, 73]. Cutaneous lipid-soluble antioxidants such as
In the last decade, endogenously produced systemic and vitamin E and squalene decay accordingly [74]. Since skin
cutaneous redox-active substances (superoxide, hydrogen surface lipids (SSL) play multiple essential roles in skin
peroxide, NO, lipid peroxides, stable end products of lipid barrier properties, skin smoothness, elasticity, and moisture,
peroxidation, oxidative metabolites of cholesterol and squa- they are regarded as natural guards of normal cutaneous
lene, etc.), previously recognised exclusively as undesirable ecology. Moreover, moderate concentrations of specific SSL
metabolic by-products and markers of oxidative damage, unsaturated components (squalene, cholesterol, and free fatty
have shown essential functions in cellular signalling and reg- acids) are able to generate oxidised lipid by-products (MDA,
ulation of cell proliferation, differentiation, migration, innate 4-hydroxynonenal, oxidised cholesterol, and others), since
immunity, energy production, ECM dynamics, vascular tone, being long recognised as key signalling molecules for skin
stress responses and adaptation, and inflammation [55, 69 immune and metabolic responses to environmental insults
71]. In this frame, the moderate plasmatic elevation of MDA and microbial invaders [70, 75, 76]. On the other hand,
(Figure 3(b)) and NO (Figure 3(a)) observed in this study excessive levels of microbially or photooxidised derivatives
may reflect the regulatory functions of these mediators both of unsaturated fatty acids and other sebum lipids could
in MCPs-induced collagen synthesis (Figure 4(a)) [69] and in induce a vicious cycle of sebum overproduction followed by
oxidation, thus maintaining inflammation characteristic for References

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Abbreviations
[7] M. Richelle, M. Sabatier, H. Steiling, and G. Williamson, Skin
MCPs: Marine collagen peptides bioavailability of dietary vitamin E, carotenoids, polyphenols,
ECM: Extracellular matrix vitamin C, zinc and selenium, The British Journal of Nutrition,
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http://dx.doi.org/10.1155/2016/1892412
Review Article
1,4-Dihydropyridine Derivatives: Dihydronicotinamide
AnaloguesModel Compounds Targeting Oxidative Stress
Astrida Velena,1 Neven Zarkovic,2 Koraljka Gall Troselj,2 Egils Bisenieks,1 Aivars Krauze,1
Janis Poikans,1 and Gunars Duburs1
1
Laboratory of Membrane Active Compounds and Beta-Diketones, Latvian Institute of Organic Synthesis, Riga LV-1006, Latvia
2
Ruer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia
Correspondence should be addressed to Astrida Velena; astrida@osi.lv and Neven Zarkovic; zarkovic@irb.hr
Received 14 August 2015; Accepted 7 October 2015
Academic Editor: Luciano Saso
Copyright 2016 Astrida Velena et al. This is an open access article distributed under the Creative Commons Attribution License,
Many 1,4-dihydropyridines (DHPs) possess redox properties. In this review DHPs are surveyed as protectors against oxidative stress
(OS) and related disorders, considering the DHPs as specific group of potential antioxidants with bioprotective capacities. They
have several peculiarities related to antioxidant activity (AOA). Several commercially available calcium antagonist, 1,4-DHP drugs,
their metabolites, and calcium agonists were shown to express AOA. Synthesis, hydrogen donor properties, AOA, and methods
and approaches used to reveal biological activities of various groups of 1,4-DHPs are presented. Examples of DHPs antioxidant
activities and protective effects of DHPs against OS induced damage in low density lipoproteins (LDL), mitochondria, microsomes,
isolated cells, and cell cultures are highlighted. Comparison of the AOA of different DHPs and other antioxidants is also given.
According to the data presented, the DHPs might be considered as bellwether among synthetic compounds targeting OS and
potential pharmacological model compounds targeting oxidative stress important for medicinal chemistry.
1. Introduction in cancer cells in which, depending on various factors, OS

may have anticancer-like effects. Its protumorigenic effects
1,4-Dihydropyridines (DHPs) [1], including Ca2+ antagonist are primarily related to induction of oxidative DNA lesions
(CA) drugs [2], are large group of structurally diverse (8-OH-G) and consequential increase of DNA mutations
compounds. Functionally, they are similar to dihydroni- that may, if not repaired, lead to genome instability and an
cotinamide redox-active synthetic compounds with radical increased rate of cellular proliferation [10]. On the other
scavenging and antioxidant (AO) properties and may be hand, antitumorigenic actions of OS have been closely linked
considered as protectors against oxidative stress (OS) and to cellular processes of senescence and apoptosis, two major
associated disorders [3]. molecular mechanisms that counteract tumor development.
Oxidative stress is extremely important for molecular Which of these two actions will dominate depends on many
pathogenesis, especially influencing the redox regulation of factors including the metabolic status of the cell, as recently
cellular signaling pathways [47]. Oxidative stress closely reviewed by Kujundzic et al., 2014 [11].
relates to presence of oxygen and nitrogen free radicals, Antioxidants (AOs) are defined as substances that, even
known as reactive oxygen species and reactive nitrogen when present in low concentrations compared to those of
species (ROS and RNS, resp.). They cumulatively increase an oxidizable substrate, prevent or significantly delay the
upon cellular exposure to various endogenous and/or exoge- oxidation process (Halliwell and Gutteridge, 1995 [12]). Their
nous insults. ROS and RNS have the two-faced character activity depends on complex factors including the nature of
and play a dual role as both deleterious and beneficial the antioxidants, the condition of oxidation, the properties
species [8, 9]. Although explored in many diseases, various of substrate oxidized, and the level of oxidation (reviewed
phenomena related to OS have been probably best studied in Kancheva and Kasaikina, 2013 [13]). Accordingly, an
antioxidative effect may be direct, resulting from direct ROS applying synthetic AO. Eventually, it was not certain also
scavenging, or indirect from the influence on various signal- that detected lipid peroxides have been generated in vivo
ing pathways related to cellular defense, that is, stress res- in the intact organs and were not artificially formed during
ponses. In relation to human physiology, antioxidants are tra- the isolation [15]. All these objections and skepticism were
ditionally classified as exogenous (supplied mostly through rejected in due time.
food) and endogenous and are further subclassified as enzy- However, some other research directions were suggested.
matic (i.e., superoxide dismutase (SOD) and catalase (CAT)) Fang et al. [18] reported two different therapeutic strate-
and nonenzymatic (i.e., glutathione, vitamins A, C, and E, gies for modulating OS in cancer and inflammation, includ-
etc.) [3]. ing (1) antioxidant therapy and (2) oxidation therapy.
DHPs could be classified as the separate group of syn- For (1), polymeric superoxide dismutase (e.g., pyran
thetic nonenzymatic, however, biomimetic AOs. copolymer-SOD), xanthine oxidase (XO) inhibitor, devel-
oped water-soluble form of 4-amino-6-hydroxypyrazolo[3,4-
2. Oxidative Stress and d]pyrimidine (AHPP), heme oxygenase-1 (HO-1) inducers
(e.g., hemin and its polymeric form), and other antioxidants
Its Prevention: Wavy Scientific Process or radical scavengers (e.g., phenolic compound canolol, 4-
DevelopmentPro et Contra vinyl-2,6-dimethoxyphenol) were used.
About (2), besides neurodegenerative diseases, cancer
There are opposite views both towards the role of oxidative
may represent yet another very interesting field for exploring
stress and about potential applications of exogenous antioxi-
antioxidants and prooxidants as therapeutic substances due
dants in onset of OS [1416].
to their cytotoxic effects (including overproduction of ROS)
Herewith, we need to mention that antioxidants have
that, if achieving proper selectivity, may be used for cancer
been studied for decades (starting from 1970s) as the tools
cells destruction (Fang et al. [18]). To achieve this goal, a
for the treatment of various disorders. The role of native and
unique therapeutic strategy was developed, named as oxida-
synthetic antioxidants (acting on lipid peroxidation (LP) in
tion therapy, by delivering cytotoxic ROS directly to the solid
biological membranes) in radiation damage and malignant
tumor or alternatively inhibiting the antioxidative enzyme
growth was seriously evaluated [17]. The overall conclusions
system, such as HO-1 in tumor. This anticancer strategy
point out antioxidants role in decreasing the damage of
was examined by use of O2 or H2 O2 -generating enzymes
cells by reducing oxidants before the occurrence of cellular
(i.e., XO and d-amino acid oxidase [DAO], resp.) and by
damage [14]. It was elicited and accented (Burlakova et al.
discovering the inhibitor of HO-1 (i.e., zinc protoporphyrin
[15]) that
[ZnPP] and its polymeric derivatives).
(i) antioxidants, nontoxic inhibitors of free radical pro- While deleterious when present at high concentra-
cesses, exhibit a wide gamut (pleiotropy) of biological tions, low concentrations of ROS exhibit beneficial proper-
activity (as further will be reported, this phenomenon ties needed for controlling physiological cellular processes
is also characteristic for the DHP antioxidants group); (reviewed in Valko et al., 2007 [19]).
Jimenez-Del-Rio and Velez-Pardo [20] have discussed
(ii) the biological effectiveness of AOs correlates with oxidative stress as an important etiopathogenic factor for
their antioxidant activity (AOA); occurrence and development of neurodegenerative diseases
(iii) depending on dose, AOs may either increase or (notably Alzheimers disease and Parkinsons disease) and
decrease the AOA; cancer. As an extension, possible preventive and therapeutic
(iv) the efficacy of AO depends on the time of introduc- values of antioxidants were also discussed. Indeed, if con-
tion in the course of medical treatment because the sidered within a narrow context of oxidative homeostasis,
development of the disease may be accompanied by antioxidants may seem to be ideal weapon in preventing
stages of changing the AOA. and fighting these diseases. However, the context of human
pathology is very broad and, so far, there was little benefit
In relation to dose-effect dependence, Burlakova et al. [15] of exogenous antioxidants in human intervention studies or
have found the nonlinear pattern: after addition of an AO, clinical trials. There are numerous reasons for these failures.
there is an initial increase of AOA, followed by returning to Maybe, the most important one is the design of the preclinical
normal and finally decreasing drastically below the normal studies, especially related to concentration of the antioxidant
value. Therefore, antioxidants may produce a specific effect used and time parameters relevant to the clinical setting
by decreasing (at low doses) or increasing (at high doses) (Kamat et al., 2008 [21]). The imbalance between uncritical
the rate of free radical reactions. Hence, the compound may acceptance of antioxidants as powerful drugs for various
be efficient AO only if it is introduced in a low dose at pathological conditions and disappointing results obtained
the stage of reduced AOA or in a high dose at the stage of in clinical studies has made a sort of confusion. This issue
AOA elevation. The widespread opinion of opponents was was addressed by Bast and Haenen [16] through listing ten
that the antioxidant function, even that of tocopherol, was misconceptions related to commercialized applications of
a side effect of its activity and important only for in vitro antioxidants: (a) pros: (1) antioxidants can cure any disease;
processes and without any role in bioobjects life. This opinion (2) the more the better; (3) any AO will do (the trick); (4)
was supported by the fact that the deficiency of natural AO AO status measures the level of health; (5) natural AOs are
tocopherol (E-avitaminosis) cannot be cured completely by superior (over synthesized ones) and (b) contras: (1) AOs
increase mortality; (2) when present at high doses, antioxi- Chemically, 1,4-dihydropyridines are synthetic hydro-
dants become prooxidant; (3) theoretically, antioxidants can- genated N-heteroaromatic compounds. They may have var-
not behave as such; (4) once used, antioxidants are inactive; ious substituents at positions 2,6-, 3,5-, and 1,4- (Figures
(5) antioxidant drugs do not work. 13). Their derivatives can be obtained synthetically in the
The first three pros clearly cross the line of realistic way Hantzsch type cyclic condensation reactions.
of thinking and cannot be considered seriously. The pro Bossert and Vater [29] postulated DHPs as a basis for
#4 was very informatively discussed by Pompella et al. [22] development of new cardiovascular drugs. Today there are
who comprehensively presented current problems with the many marketed drugs which contain 1,4-DHP ring as basic
methods (ORAC, oxygen radical absorbance capacity; ferric- scaffold [3032] (Figures 1 and 2).
reducing ability of plasma; and TEAC, Trolox equivalent Grover et al. [33] classified dihydropyridine nucleus (skel-
antioxidant capacity) routinely used for measurement of total eton) as a novel pharmacophore and offered some examples
antioxidant capacity (TAC) in plasma (Pompella et al., 2014 related to DHPs pleiotropy. So far, AOAs have been revealed
[22]). These include lack of needed specificity, especially rel- for several groups of DHP compounds and DHP-based drugs
evant for ORAC related measurements. Instead, precise mea- [3436], contributing to their well-known pleiotropic ways of
surement of specific compounds is recommended. Regarding action (antiaging, neuroprotective, anticancer, antibacterial,
the pro #5, the situation does not seem entirely clear, [37] and many more). These features are promising for
as some published metastudies related to protective role of development of novel drugs in the future [32, 38].
vitamin C in coronary heart disease showed some contra- It is well known that hydrogen donors such as amines,
dictions (better protection with dietary vitamin C versus thiols (aminothiols), or phenols (plant phenols and polyphe-
synthetic vitamin C) (Ye and Song [23]; Knekt et al. [24]). nols as well as synthetic hindered phenols) act as antioxidants,
In any event, this kind of research is anything but simple, as primarily through inhibition of oxidation reactions of various
observed health effects of fruit and vegetable ingestion are chemical targets/substrates. Similarly, depending on their
certainly related not only to the content of vitamin C but particular chemical structure, 1,4-dihydropyridines have sig-
also to other macro- and micronutrients and phytochemi- nificant hydrogen donor ability (see further in Section 3.2).
cals, proven to confer additional health benefits (Carr and This feature allows them to act as direct inhibitors of free
Vissers [25]). Similar to pros, stated contras seem to be radical reactions. It further classifies them as specific group of
a common misconcept related to the design of the study (this dihydropyridine type of antioxidants. However, under certain
is especially relevant for epidemiological studies), relevance conditions, primarily dependent on individual structure and
of a specific pathological condition and measurement of its applied dose, DHPs can act as prooxidants (see further in
outcomes, and, finally, complexity of a living organism. For Section 3.8).
all these reasons, there is the realistic need for well-designed
On the other hand, some DHPs may exert synergistic
epidemiological, clinical, and molecular studies that would
effects when applied together with other types of AOs [39].
offer firm evidence and undoubtful conclusions on the role of
antioxidants on human health (see also Sections 3.8 and 3.9). They can also be involved in the redox regulation of Ca2+
There are still unanswered questions related to oxidative ion channels [40]. Namely, oxidative stress, characterized by
stress and its mediators in pathogenesis of OS-associated significant increase of ROS, closely relates to cellular imbal-
diseases. However, it is clear that overproduction of ROS ance of Ca2+ ions. Such a CA activity of DHPs can also result
has harmful cellular effects. For that reason, small synthetic in the indirect OS modulation as an additional positive side
antioxidants, molecular scavengers, have been developed to effect. Accordingly, DHPs, acting as CA and as antioxidants,
be used in various pathological conditions. The first one, may modify various OS-associated pathological processes by
implemented in the clinic for acute brain infarction, was influencing cellular redox signaling potential. Additionally,
3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186, Edaravone, multiple biological effects of DHPs attenuating OS could be
Radicut, norphenazone), approved until now only in Japan important at drug-drug interactions by combination therapy
(Tabrizchi, 2000 [26]). So far, its free radical scavenging using DHPs and other CA and/or antioxidants.
properties were revealed by various biological effects (antiox- It should be mentioned that the studies on the possible
idant, attenuation of cytokine production, antiapoptotic, AOA of 1,4-DHPs have begun due to the assumption that
antinecrotic, and some other effects), as recently reviewed these substances could be useful for the design for novel
(Kikuchi et al. [27]). antioxidants intended to be used primarily in the food tech-
nology, notably as animal chow stabilizers [4143]. The AOAs
3. 1,4-Dihydropyridines: A Separate of 1,4-dihydropyridine derivatives, 2,6-dimethyl-3,5-dieth-
Group of Bioantioxidants oxycarbonyl-1,4-dihydropyridine (Hantzsch ester (HEH),
diludine) and its close analogues, 4-unsubstituted 1,4-DHPs,
1,4-Dihydropyridines could be used as model compounds for were discovered by Latvian scientists that intended to use
studying molecular mechanisms of action modulated by cel- them for the termination of the lipid peroxidation (LPO) in
lular enzymes NADH and NAD(P)H due to their structural various chemical lipid substrates/mixtures target (solutions,
analogy to 1,4-dihydronicotinamide [28]. This structure rep- emulsions, and liposomes) [44, 45]. Afterwards, antioxi-
resents the active part of these reduced coenzymes, which are dant properties of several calcium antagonists DHPs were
important modulators of various enzymatic redox reactions discovered [31, 4653]. Interestingly, research on the AOA
and are involved in electron transfer. of DHPs on LPO continues nowadays, including several
NO2 NO2
C2 H5 OOC COOC2 H5 H3 COOC COOCH3 H3 COOC COOCH2 CH(CH3 )2
H3 C N CH3 H3 C N CH3 H3 C N CH3

H H H
Diludine Nifedipine Nisoldipine
(Hantzsch ester, diethone, HEH)
Cl
CF3 Cl Cl
H3 COOC NO2 H3 COOC COOC2 H5 H3 COOC COOC2 H5
H3 C N CH3 H3 C N CH3 H3 C N CH2 OCH2 CH2 NH2

H H H
Bay K 8644 Felodipine Amlodipine
NO2 NO2
PhHC=HCH 2 COOC COO CH2CH2 OCH3 (H3 C)2 HCOOC COO CH2CH2 OCH3
H3 C N CH3 H3 C N CH3
H H
Cilnidipine Nimodipine
NO2
OCHF2 OCHF2
C3 H7 OH2 CH2 COOC COOCH2 CH2 OC3 H7 H3 COOC COOCH3 H3 COOC COOC2 H5

H H H
Cerebrocrast Foridone Nitrendipine
(ryodipine)
H3 CO C(CN)CH2CH2CH2N(CH3)CH2CH2 OCH3
CH(CH3) 2
H3 CO OCH3
Verapamil
Figure 1: Structures of the most known 1,4-dihydropyridine derivatives and some non-DHP Ca2+ antagonists.
interdisciplinary projects funded by EU, in particular the and published between 1986 and 1990 are summarized in the
COST B35 action [51, 52]. review of Sausins and Duburs [57]. In 1993, Kazda [58] has
reviewed twenty years of dihydropyridines, including their
3.1. Synthesis of 1,4-Dihydropyridines: Routes and Approaches. synthesis, chemistry, progress in pharmacology, and therapy,
Classical 3-component Hantzsch synthesis of DHP com- and some other applications. Since then, there were many
pounds [5457] is usually performed in solutions (including important discoveries in this field and there is a time for
ionic liquids) by heating. Discoveries related to this process a review on another twenty years of DHPs. It has to be
NO2 NO2
H3 COOC COOC(CH3 )2 CH2 N(CH3 )CH2 CH2 CHPh2 H3 COOC COOCH2CH2 N(CH3 ) CH2 Ph
H3 C N CH3 H3 C N CH3
H H
Lercanidipine Nicardipine
N Cl
O
N Cl COOC(CH3 )3
H3 COOC COOCH(CH3 )2 H3 COOC COOC2 H5 C2 H5 OOC COOC2 H5

H H H
Isradipine Felodipine Lacidipine
NO2 NO2 NO2
(H3 C)2 HCOOC COO N CHPh2 H3 COOC COO(CH2 )7 CH3 H3 COOC COO
N
CH2 Ph
H3 C N NH 2 H3 C N CH3 H3 C N CH3
H H H
Azelnidipine Bay O 5572 Benidipine
NO2 NO2 NO2
H3 COOC COOCH2CH2 N N CHPh2 H3 COOC COO H3 COOC COOC(CH3)3

N
CH2 Ph
H H H
Manidipine Barnidipine Mebudipine
OCH3 O
NO2
O(CH2 )3 N(CH3 )CH2 CH2 O O
S
S
OCOCH3 H3 CO H3 COOC COOCH(CH3)2
H
N
O O N
NC N CH3
CH 2 CH2 N(CH3) 2 CH3 H
Diltiazem Semotiadil Nivaldipine
Figure 2: Structures of the most known 1,4-dihydropyridine derivatives and some non-DHP Ca2+ antagonists.
mentioned that nearby this classical multicomponent synthe- various databases (see http://www.organic-chemistry.org/
sis also a process to obtain structurally diversified 1,4-dihyd- namedreactions/hantzsch-dihydropyridine-synthesis.shtm
ropyridines at sophisticated conditions was recently reviewed [60]). For example, http://www.scifinder.com/ [1] database
by Wan and Liu [59]. lists approximately 1000 citations on the simple DHP com-
Many discoveries relevant for novel routes in DHP pound, diludine. Reaxys database [61] contains data related
designing and synthesis were published and deposited in to variations in starting materials, intermediates as building
Cl
O O Cl O OCHF2 O OCHF2
CN CN CN CN
H3 C H3 C H3 C H2 N
H3 C N SCH3 H3 C N SCH3 H3 C N SCH3 H3 C N SCH3

H H H H
OSI-1146 OSI-3761 OSI-9642 OSI-3701
Figure 3: Molecular structures of OSI-1146, OSI-3701, OSI-3761, and OSI-9642 (according to [146]).
blocks, media, and reactions routes. Water and ionic liquids Hantzsch esters have been extensively utilized as stoichio-
as reaction media, microwave and infrared irradiation, new metric biomimetic reducing agents. Recent summarized lit-
catalysts, solid phase synthesis, and biotechnology based and erature about DHPs as reducing agents, including references
green chemistry approaches were also proposed as attractive on diludine, may be found on specialized websites: http://
options for syntheses of DHPs [6266]. www.organic-chemistry.org/chemicals/reductions/ [75].
Furthermore, several new dihydropyrimidin-(2H)-ones DHPs form free radicals in chemical, electrochemical,
(DHPMs), close analogues of DHPs, were prepared in the and biological oxidation processes. The kinetic parameters
Biginelli reaction under ultrasound irradiation and in the and pathways of decay of the cationic radicals formed as
presence of NH4 Cl. Some of these compounds, when tested primary products in the course of electrooxidation of the
in vitro at concentrations higher than 100 M [67], showed esters of 1,2- and 1,4-dihydropyridine have been extensively
AOAs, manifested as inhibition of LPO induced by complex studied [76].
Fe + EDTA and reduction of ROS levels. The regenerative system of nicotinamide cofactors may
Recently, Sun et al. [68] reported about the synthesis and involve oxidizing or reducing reagents, regulating enzymes,
antioxidant activity of a series of novel 3-chalcone-substituted and photochemical reactions. Thus, in situ regeneration
1,4-dihydropyridine derivatives, based on dimethyl or diethyl of the consumed cofactors was observed in the biosys-
2,6-dimethyl-4-phenyl-1,4-DHP-3,5-dicarboxylate. tems engineering, which create superior biocatalysts by the
reduction of NAD(P)+ , which can lead to the 1,4-DHP
3.2. 1,4-Dihydropyridines as Hydrogen Donors. Steric, elec- product (which is the only active form) and to the 1,6-
trostatic, and hydrophobic descriptors in DHP molecule DHP compound [77]. The NADPH models of HEHs can be
could serve as its potential pharmacophores [2]. In case regenerated in situ as biomimetic hydrogen sources by means
of Hantzsch ester this implies partly hydrogenated N- of transition metal/Brnsted acid catalyzed relay asymmetric
heteroaromatic DHP nucleus itself or its fragments, that is, hydrogenation [78]. General regeneration strategies were
NH group or C-4 H- atom, as hydrogen donors necessary reviewed by Chenault and Whitesides [79]. Based on these
for the AO activity and/or carboxylic ester side groups (its strategies, particularly related to methods of preparation and
C=O group and O atom as hydrogen bond acceptors) in practical use of esters of 2,6-dimethyl-1,4-dihydropyridine-
positions 3- and 5- and alkyl side groups in positions 2- 3,5-dicarboxylic acid as antioxidants that might be probably
and 6- (as hydrophobic features) (Grover et al. [33] and applicable for radioprotection and adjuvant treatment against
Tikhonov and Zhorov [69]). The presence of labile hydrogen metastases, several patents were prepared [80].
atoms (mainly in positions 1,4-) in DHPs molecule assigns Sambongi et al. [81] have found that the novel water-
significant hydrogen donating ability to these compounds. soluble Hantzsch 1,4-dihydropyridine compound (the potas-
DHPs (2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxy- sium salt of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicar-
lic acid esters) can be oxidized in chemical (Dubur and boxylic acid monomethyl ester) functions in biological pro-
Uldrikis [70]), electrochemical, enzymatic (Duburs et al. cesses through regeneration of NADH. Various parameters
[71]), and biological (including metabolism and biotransfor- related to nicotinamide coenzymes regeneration, especially
mation) systems. As already stated, dihydropyridines (espe- in a light of chiral compounds, have been published recently
cially unsubstituted in position 4) may transfer the hydro- [82], while Okamura et al. [83] reported the use of the
gen, similar to the reduced diphosphopyridine nucleotides, oxidative conversion of dihydropyridine to pyridinium ion
NADH and NADPH (Scheme 1) (Mauzerall and Westheimer and the metabolic trapping principle as an approach for
[28]), while HEH hydrogen transfer studies and search for measuring in vivo cerebral redox states.
novel NADH model compounds are continuously developing
(Xie et al. [72]). 3.3. Antioxidant Activity (AOA) and Antiradical Activity
Tamagaki et al. [73] observed metal-ion-facilitated oxida- (ARA) of 1,4-Dihydropyridines. Antioxidative activity of 1,4-
tions of DHPs with molecular oxygen and hydrogen peroxide. DHPs was first evaluated and studied in the Latvian IOS
On the other side, Tirzite et al. [74] studied some 1,4- (Tirzit and Duburs [39], Zilber et al. [44], and Dubur et al.
DHP derivatives as reductants in relation to trivalent iron. [45]).
R (H)
O H R O O O O R (H) O
+
e H e
e H
H H+
R=H
N N N
R R
Scheme 1: Reactions of 1,4-dihydropyridines leading to the formation of pyridine derivatives.
In the field of ARA, pioneering work was made by Schel- The activity against DPPH radical was found for the
lenberg and Westheimer [84] in 1965. In 1979, Schellenberg 5-acetyl-2-alkylthio-4-aryl-6-methyl-1,4-dihydropyridine-3-
[85] revealed the free radical oxidation of a dihydropyridine carboxylic acid nitriles [97], structural analogues of the
following Huyser et al. [86] who reported hydroxyl radical 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-DHP-carbonitrile
quenching by DHPs, especially Hantzsch ester, studying free (studied as mitochondriotropic compounds; see further in
radical oxidation of DHPs in vitro, in the Fenton system. the text, Section 3.3.1 (2)-(b)). The highest antiradical activity
AOA and ARA of various 1,4-DHPs were further studied occurred for a compound which contains two hydroxyl
by several different methods in both in vitro and ex vivo/in groups in the 4-phenyl substituent.
vivo systems [3, 31, 4453]. CA nisoldipine, nimodipine, DHPs were proved to decrease oxygen uptake (2-3-
nitrendipine, nifedipine, and nicardipine have AOA that fold) in the heme (methemoglobin, hematin, hemin, and
correlates with their lipophilicity (modified Buege and Austs cytochrome C) catalysis by oxidation of emulsions of esters
method of TBA determination, applied in model of rat brain of unsaturated fatty acids and liposomes of phospholipid
cortex ischemia/reperfusion) [87]. phosphatidylcholine (Zilber et al. [44] and Dubur et al. [45]).
N-Aryl-DHPs, designed as sirtuin activators, were further Reactivity of nitrosoaryl and nitroaryl derivatives of 1,4-
reported as suitable agents for neuroprotection due to their DHPs toward alkyl, alkylperoxyl radicals, and ABTS radical
radical avoidance properties (Hardeland [88]). cation was found in various LP modeling systems suitable
1,4-DHPs inhibit free radicals and, consequentially, the for determination of DHPs AOA and ARE features [98
cascade of events related to lipid peroxidation. They may 104]. Diludine and foridone and its analogues were shown to
influence several stages (initiation and/or propagation) of the inhibit lipid peroxidation through inhibitory effect on lipoxy-
lipid peroxidation cascade, which consist of 10 reactions genase, in emulsions and in reversed micelles (Tsetlin et al.
[89] (detailed discussion in Section 3.3.1 (2)-(b), Scheme 2). [105] and Panek et al. [106]). In addition to inhibition of ther-
However, considering the great number of AO com- mally initiated oxidation of methyloleate in the solution [107]
pounds (including DHPs) and the diversity in their action (where AOA of 4-unsubstituted 3,5-dicarbonylderivatives
mechanisms [90], in vivo studies are not always convincing of 2,6-dimethyl-1,4-DHPs is not linearly dependent on
and conclusive. Therefore, concise in vitro models are neces- the inhibitor concentration), DHPs derivatives containing
sary to screen each compound with antioxidative properties. hydroxy, alkoxy, or dimethylaminophenyl substituents in
Antioxidants are designed to react readily with oxidizing position 4 were shown to prevent loss of -carotene in the
species and are often extensively oxidized already during disperse system of -carotene and methyllinoleate (Plotniece
incubations at atmospheric oxygen tension (oxidation of et al. [108]).
some water-soluble DHPs in water (buffer) solutions is very The AOA of DHPs has been detected using different
fast, especially in the presence of light). Even during a methods in various systems where lipid free radical gen-
relatively short incubation period, the concentration can drop eration (nonenzymatic, Fe2+ -dependent, and/or enzymatic,
drastically, and the real potency of the compound could be NADPH-dependent) took the place [109112]. This activity
underestimated [90]. was further confirmed in vivo, through prevention of damage
caused by renal ischemia and reperfusion, as shown for
3.3.1. Common AOA and ARA Features of Some DHPs diludine [113].
Some redox properties of calcium antagonist dihydropy-
(1) In Vitro (in Solutions, Emulsions, and Liposomes). Basic ridines were revealed through electroanalytical studies [114].
molecular principles related to antioxidative and antiradi- Competitive kinetic procedure was used for exploring the
cal activity of various antioxidants, including DHPs, were AO capacity of five (four 1,4-DHPs: lacidipine, felodipine,
published recently [91]. These data show that DHPs react nifedipine, and amlodipine, and one 1,2-DHP compound
with various types of free radical species, stable free radicals GR44966) CA and one calcium ion agonist (Bay K 8644). All
(DPPH a.o.) and alkyl radicals and with oxygen and nitrogen but one (amlodipine) antagonist displayed an unambiguous
free radicals. Some derivatives of DHPs may quench a singlet AO capacity (crocin test). The calcium agonist DHP revealed
oxygen and may react with peroxynitrite anion [9295]. no reaction with peroxyl radicals. Lacidipine was the most
Reactivity of DHPs toward alkyl radicals was studied effective. A calcium agonist Bay K 8644 is quite resistant to
electrochemically [96]. oxidation and does not bind H+ . This could be important
fact in the interaction with the target proteins (it should be and LDL antioxidant activity) effect [120]. It includes a
mentioned that there are no studies on LP with other Ca2+ combination of peroxide-degrading and peroxyl radical scav-
agonists). enging reactions, thus demonstrating the importance of LP
The decreased oxidation potential correlates with AO during LDL oxidation and cytotoxicity induced by oxidized
capacity and increased basic character. These findings suggest LDL. Cytoprotection is associated with inhibition of oxidant-
the relevance of the electron density on the DHP ring. induced increases in intracellular free calcium.
For all the DHP compounds investigated, the overall Similar to the other model systems, the recorded values of
oxidation process proceeds through two consecutive one- the tested DHPs related to AO activity on LDL LP and related
electron releases: a primary one-electron step accompanied events [119131] depend on the prooxidant model system and
by a fast proton release and the formation of a neutral radical methods used for activity measuring (see Tables 15).
(PyH ) undergoing a second, much easier one-electron step Commercial Ca2+ antagonists (including 1,4-DHP deri-
[114]. vatives), as well as some other 1,4-DHPs with less CA activity,
The final product is the protonated form of the parent were shown to decrease the rate of oxidation (detected as
pyridine derivative. This pattern is relevant for the antioxida- TBARS) of low-density lipoprotein (LDL) induced by Cu2+
tive activity, since the radical intermediate is far less prone to ions (CuSO4 ) in two different cell lines: U937 human mono-
be reduced than oxidized. cyte-like and J774A.1 murine monocyte-macrophage cell
In the case of CA DHPs, the release of protons compli- line (Rojstaczer and Triggle [119]). The strongest effect was
cates the overall oxidation process by introducing a para- recorded for vitamin E, followed by felodipine, 2-Cl analogue
sitic side reaction where a coupling between protons and the of nifedipine, nifedipine, amlodipine, nitrendipine, vera-
starting species takes place. pamil, and diltiazem.
This DHP self-protonation subtracts part of the original Rojstaczer and Triggle [119] found that CA from different
species from the electrode process because the parent cationic chemical groups had a concentration-dependent effect as
species are no longer electroactive. antioxidants against LDL oxidation (see Table 1). However,
Conversely, the calcium agonist DHP, which is less prone the order of potency (activity rank order, ARO) of the drug(s)
to be oxidized, turned out to be so weak base to be even again depends on the oxidation system and the antioxidant
unable to undergo the self-protonation reaction. assay. Both CA and antioxidative effects relate to the 2- (or o-,
Thus, the combined effect of oxidation potentials and orto-) substituent of the 4-phenyl ring in the same potency
proton binding capacity of DHPs is a key element for the order > [119]. On the other hand, the require-
redox transition, relevant for their AO activity. Yet, opposing
ment for the 1,4-DHP ring is essential for both AOA and Ca2+
effects (antagonistic versus agonistic) on protein targets as
channel antagonism. A charged substituent at the position C-
calcium ion channels connected with protein thiol oxidation
2 of the 1,4-DHP ring influences the AO activity (analogous
to disulfide should be also considered [114].
to [4653]). However, some other factors should not be
Kourimska et al. [115] found AO effect of diludine (HEH)
neglected: for example, although amlodipine has a positively
in edible oil. Reactivity of 1,4-DHPs toward SIN-1-derived
charged amine at this position, this modification makes it less
peroxynitrite was shown by Lopez-Alarcon et al. [116]. Olek
lipophilic and, indirectly, less potent antioxidant.
et al. [117] discovered antioxidative activity of NADH and its
analogue in vitro. Similar results were obtained when testing antioxidant
Further see, as referred in several subparts below, Sections effect of CA on LDL peroxidation in bovine aortic endothelial
3.4, 3.5, and 3.7. cells (BAECs) (Cominacini et al. [123]; see Tables 2 and 3) as
well as in HUVECs (Lupo et al. [129]) (see Table 4).
(2) Ex Vivo (on Lipid Peroxidation in LDL, Mitochondria, Cominacini et al. [123] observed antioxidant effect of
Microsomes, and Cells). Main chemical structures of DHPs CCBs and -tocopherol in BAECs. The order of potency
examined in numerous studies and reviewed in this paper are (see Tables 2 and 3) [123] was however different than in
presented in Figures 1 and 2. U937 human monocyte-like and J774A.1 murine monocyte-
macrophage cells (see Rojstaczer and Triggle [119], Table 1).
(a) Various DHPs: Calcium Antagonists as Inhibitors of LDL The tested DHPs were lacidipine, amlodipine, lercanidipine,
Peroxidation. Free radicals induce peroxidation of LDL. nimodipine, and nifedipine (in two different intracellular
This process proceeds by a chain mechanism which reveals concentrations: 2 and 4 fmol). ROS production was signif-
phosphatidylcholine hydroperoxides and cholesteryl ester icantly lowered only by lacidipine (which is the compound
hydroperoxides as the major primary products [118]. Calcium with the highest lipophilicity) and lercanidipine; the effect
antagonist DHPs could act as antioxidants on LDL at least of lacidipine was much more evident than lercanidipine.
in three ways: (1) as inhibitors of isolated LDL peroxi- Surprisingly, amlodipine, nimodipine, and nifedipine had no
dation, caused by various inducers (Cu2+ ions, UV light, effect on ROS formation suggesting that the positive effects
and xanthine/xanthine oxidase system); (2) if preincubated on the earliest events of atherosclerosis are a peculiarity of
with cells, preventing against intracellular LDL oxidation; (3) lacidipine molecule through its antioxidant activity.
preventing against the harmful effect of oxidized LDL on cells The strong AO action of lacidipine may be related to
and decreasing cytotoxicity [119131]. the lipophilic cinnamic acid side chain, which favors a drug
Combined application of ascorbic acid and CA DHPs partitioning in the membrane due to favorable physicochem-
(amlodipine and felodipine) has an additive (cytoprotective ical (hydrophobic) interactions of drug hydrophobic residues
Table 1: Relative structure-function relationships of calcium antagonists (DHPs, verapamil, and diltiazem) and vitamin E. Effect on oxidative
modification of isolated ex vivo human low-density lipoprotein using two various oxidation systems (copper (II) ions induced and monocyte
induced). Compiled according to data presented by Rojstaczer and Triggle [119].
Systems of LDL oxidation

Copper (II) ions induced system (comparison of three methods) Monocyte induced cell
oxidation system
Compound Methods
Degradation of oxidized Relative electrophoretic
Reduction of TBARS level of LDL TBARS content of LDL
[125 I] LDL by J774 mobility of LDL on
(relative efficacy) (in %%)
macrophages agarose gel
Relative efficacy (activity rank order (ARO); ARO = I for the most effective); effective inhibitor concentration [IC], in M
++ ++ 25 M 25 M
Amlodipine
(ARO = IV) (ARO = IIV) 50 M (ARO = IIIV)
+++
+++++ 25 M, 65 9%
Felodipine (ARO = I) 50 M
(ARO = I) (ARO = II)
25 M, 97 2%
+++ ++ 25 M, 96 2%
Nifedipine 10 M; 50 M
(ARO = III) (ARO = IIV) (ARO = I)
2-Chloro
++++
analog of
(ARO = II)
nifedipine
4-Nitro
++ 25 M
analog of
(ARO = IIV) (ARO = IIIV)
nifedipine
++
Nitrendipine No effect
(ARO = IV)
++ ++ 25 M
Verapamil
(ARO = IV) (ARO = IIV) (ARO = IIIV)
+
Diltiazem No effect
(ARO = V)
-Tocopherol ++++++ 1 M; 5 M; 10 M;

(vitamin E) (ARO = I) 50 M
Table 2: Reduction of intracellular ROS in BAECs by CA DHPs. Table 3: Modulation of ROS formation in BAECs by CA (DHPs and
Compiled according to data reported by Cominacini et al. [123]. verapamil) and vitamin E. Compiled according to data presented by
Cominacini et al. [123].
Cellular amounts of compounds (in fmol/cell)
Compound determining the 50% reduction (IC50 ) in Method of flow cytometry (reduced
intracellular ROS concentrations 2 ,7 -dichlorofluorescein diacetate
Lacidipine 4.6 0.7 (DCFH-DA) oxidation by ROS)
Compound Activity rank order
Lercanidipine 9.2 0.7
(ARO = I for the highest activity; ARO = III for
Amlodipine 15.3 0.8 the mindest activity)
Nifedipine 16.4 0.7 (Effective [IC]: 1; 5; 10; 50 M)
Nimodipine 17.2 0.9 Lacidipine + + + (ARO = I)
Lercanidipine + + (ARO = II)
Amlodipine No effect
with polyunsaturated groups of membrane phospholipids.
Nifedipine No effect
However, DHPs can also reduce the oxLDL-induced ROS
concentration by affecting some intracellular ROS producers, Nimodipine No effect
such as NADPH oxidases, xanthine oxidase, and cyclooxyge- Verapamil + (ARO = III)
nase enzymes. The activity of these enzymes contributes to -Tocopherol + + + (ARO = I)
intracellular ROS elevation [125].
Preincubation of HUVECs with lacidipine inhibited an
increase of intracellular ROS caused by oxidized LDL [124].
Lupo et al. [129] have studied the dose-dependent (1, 5, against normolipidemic human blood LDL oxidation com-
10, and 50 M) AOA of various CA (verapamil, diltiazem, pared with -tocopherol by measuring the content of TBARS
and DHPs: nifedipine, amlodipine, isradipine, or lacidipine) and the diene formation (see Table 4).
Table 4: Normolipidemic human blood LDL (0.25 mg/mL) in vitro oxidation in the presence of 5 M CuSO4 and CA of 3 types (DHPs,
verapamil, and diltiazem) and vitamin E. Compiled according to Lupo et al. [129].
Methods
TBARS method (fluorimetry at 515 nm/533 nm, 4 hours
Inhibition of conjugated diene formation (at 234 nm)
preincubation of LDL with compounds and copper (II)
expressed as prolongation of induction period (in %%
ions; 320% TBARS increase in control during 4 h
Compound of control). contr = 36.8 min.
period)
Activity rank order (ARO Activity rank order (ARO
Effective [IC] (in M): = I for the highest activity; Effective [IC] (in M): = I for the highest activity;
1 M; 10 M; 50 M ARO = VII for the 1 M; 5 M; 10 M; 50 M ARO = VII for the
mindest activity) mindest activity)
5 M;
10 M;
Nifedipine ARO = III 10 M, 150%; ARO = III
50 M
50 M, 213%
5 M;
Amlodipine 50 M ARO = IV 10 M, 122%; ARO = IVVI
50 M, 138%
10 M, 150%;
Isradipine 50 M ARO = VI ARO = IVVI
50 M, 183%
1 M; 5 M;
Lacidipine 10 M; ARO = II 10 M, 192%; ARO = II
50 M 50 M, 283%
10 M, 150%;
Verapamil 50 M ARO = V ARO = IVVI
50 M, 178%
Diltiazem No effect No effect (ARO = VII) No effect No effect (ARO = VII)
1 M; 5 M;
Vitamin E 10 M (IC50 ); ARO = I 10 M, 230%; ARO = I
50 M (20% of control) 50 M, 370%
Table 5: Antiproliferative effect (oxLDL-induced HUVSMCs proliferation) of CA DHPs and simultaneous oxLDL-induced ROS production
scavenging. Comparison with N-acetyl-L-cysteine, NAC (intracellular ROS scavenger). Compiled according to data presented by Zou et al.,
2012 [130].
Methods
Antiproliferative effect against proproliferative effect
oxLDL-induced ROS production (fluorescent DCF
DHP compound induced by oxLDL (50 g/mL) (UV detection of
(2 ,7 -dichlorofluorescein) production)
formazan production from tetrazolium salt)
Effective [IC] in M and I in %
No effect
Amlodipine 3 M I = 18% 3 M; 10 M
I = 20%
S()-Amlodipine No effect No effect
10 M; I = 21%
Lacidipine 10 M I2/3 of control
30 M I = 27%
N-Acetyl-L-cysteine, 5000 M
I = 28%
NAC (5 mM)
As presented (Table 4, according to [129]), for diltiazem a possible antiatherogenic mechanism of CA, since oxidative
(poor lipid solubility), no AO was detected, whereas the other modification enhances the atherogenic potential of LDL.
CA and -tocopherol have demonstrated AOA at least at The lipid peroxidation of LDL, promoted either by UV
concentrations of 10 and 50 M: -tocopherol > lacidipine radiation or by copper ions, was inhibited (antioxidant effect)
> nifedipine > isradipine, verapamil, and amlodipine. Addi- by nisoldipine in a dose-dependent manner (IC50 values were
tionally, -tocopherol and lacidipine were able to significantly evaluated at around 10 M), nimodipine was less potent (IC50
attenuate in vitro LDL oxidation at 1 and 5 M. These around 50100 M) and nicardipine almost inactive. In addi-
results have confirmed the highest activity for the strongly tion to this indirect protective effect, CA DHPs nisoldipine
lipophilic DHP type CA compound lacidipine. This might be and nimodipine exerted direct protective effect on lymphoid
cells, against toxicity of previously oxidized LDL. The IC50 fluorescence analysis, AGE-ELISA, quantitation of lipid per-
values were 6 2 and 80 20 M, respectively [122]. The oxidation, and TBARS measurement of long-term g/go LDL.
inhibition of the cytotoxic effect of LDL oxidized in the The effects of lipophilic DHP calcium channel blockers
presence of DHP type Ca2+ channel blockers correlated well on oxidized LDL-induced proliferation and oxidative stress
with protection from oxidation by these compounds. Com- of vascular smooth muscle cells were also studied [130] (see
plete protection cannot be obtained because the DHPs are Table 5).
cytotoxic themselves. The potential relevance to the preven- Lacidipine and amlodipine reduced carotid intima-
tion of atherogenesis is envisaged. media thickness by decreasing proliferative effect of oxLDL,
DHP type CCB nifedipine was the most effective inhibitor whereas (S-)-amlodipine had no antiproliferative effect. ROS-
of oxidation promoted either by UV radiation or by copper MAPKs (mitogen-activated protein kinases) pathway might
ions in experiments with cultured lymphoid cells LDL (2 mg be involved in the mechanism.
apoB/mL); CCBs from other two CCB classes, diltiazem and Both 1,4-DHP CCBs lacidipine and nifedipine reduce
verapamil, were only poorly active or completely ineffective plasma and LDL oxidation and formation of oxidation-
[121]. The protective effect of nifedipine occurs at two levels: specific epitopes. Their application may also relate to pro-
besides its direct antioxidant effect by inhibition of LDL longed survival of rats, independently of blood pressure mod-
oxidation, it also exhibits a direct cytoprotective effect against ifications (in the SPSHR model, 1 mg/kg per day lacidipine
cytotoxicity of oxidized LDL by yet unknown mechanisms. and 80 mg/kg per day nifedipine). These results suggested
The protective effect of CCBs was not due to an inhibition that the protective effect of these two 1,4-DHP drugs in
of LDL uptake. This effect seems to be independent of the vivo, as shown in cerebral ischemia and stroke, may in part
inhibition of LDL oxidation per se since LDL was oxidized result from inhibition of LDL oxidative process, although
in the absence of the drug before the incubation with these two drugs possess different lipophilic properties [131].
cells. Moreover, this direct protective effect was observed Both lacidipine (0.3 and 1.0 mg/kg) and nifedipine (80 mg/kg)
at lower concentrations (IC50 of 1 0.2 M) compared to prolonged lag time of the conjugated diene formation in
the antioxidant effect (IC50 of TBARS inhibition is around LDL isolated from arterial wall, and max . These drugs
10 2 M at UV promoted and 4 0.5 M by Cu2+ ions significantly reduced electrophoretic mobility of oxLDL from
initiated). The AO effect of nifedipine is also correlated with SPSHR subjected to X/XO oxidation system. 1,4-DHP CCBs
the protection of endogenous tocopherols (IC50 = 50 M). also protected apolipoprotein B, which is important for the
It was suggested that the AO effect of CCBs protected cells binding with macrophage LDL receptor lysine residues. The
indirectly from the cytotoxic effect of oxidized LDL [121]. doses used (>106 mol/L for SPSHR and normotensive WKY
A recent study has reported that beneficial vascular effects
rats), however, are 2 to 3 orders of magnitude higher than
of lercanidipine in diabetic rats depend on its antioxidant
those inhibiting vascular smooth muscle contraction in vitro
activity related to attenuating the increase in oxidative
and in vivo. They also exceed values that are commonly
stress and in vascular matrix metalloproteinase-2 (MMP-
used in clinical practice. The daily dose of lacidipine for
2) (Martinez et al. [126]). Lesnik et al. [127] studied the
hypertensive patients is 0.07 mg/kg, 4- to 14-fold lower than
impact of a combination of this calcium antagonist and a
the 2 doses used in SPSHR. The maximum daily dose of
-blocker atenolol on cell- and copper-mediated oxidation
nifedipine given to hypertensive patients is 2.0 mg/kg, 40-
of LDL and on the accumulation and efflux of cholesterol
fold lower than what were used [131]. These discrepancies may
in human macrophages and murine J774 cells. They realized
be related to differences in bioavailability of CA between rats
that lercanidipine reduced the oxidative modification of LDL
and humans [131].
rather than diminished cholesterol accumulation in human
foam cells. Accordingly, in routine clinical use, 1,4-DHP CCBs do not
Comparing the antioxidative action of CA (DHPs, amlo- reach the concentrations required for antioxidant activity in
dipine, lacidipine, nifedipine, and isradipine, as well as diltia- vitro [131].
zem and semotiadil) in the copper-catalyzed oxidation of Another data concerning the effect of CA DHPs on OS
low-density lipoprotein (LDL) with that of glycated (g)/gly- related to LDL is presented under Section 3.5.
coxidated (go) LDL demonstrated that the strongest AO
effects during long-term LDL glycation are seen for isradip- (b) Effect of DHPs on Isolated Rat Liver and Heart Mitochon-
ine, lacidipine, nifedipine, and semotiadil [128]. Inhibitory dria. As a major cellular source of oxygen radicals (Cadenas
effects were in the range 105 103 M. Authors suggested that, [4, 5]), mitochondria are promising targets for pharmaco-
due to the increased generation of ROS by glucose-modified logical and toxicological actions of various membrane-active
LDL, the chain-breaking capacity of CA may be overridden. compounds, including several 1,4-DHP derivatives. Zernig et
The AOA of CA depends on their lipophilicity and their al. [132] have discovered CA binding sites associated with an
ability to incorporate into the LDL particle, that is, to reach inner mitochondrial membrane anion channel.
the site of peroxidation. CA, like other AOs, significantly More than 40-year long research on mitochondrial effects
retards advanced glycation end products (AGE) formation, of the DHPs (on their bioenergetics, chemiosmotic proper-
whereas initial glycation reactions, such as Amadori product ties, and ion fluxes) clearly points them out as mitochondri-
formation, are only weakly inhibited. The observation that otropic compounds.
both oxidative changes and at least long-term glycation effects The activity of the first 35 synthesized compounds
are indeed drastically reduced by CA is corroborated by (derivatives of 1,4-DHP, their heteroaromatic analogues,
NAD-H+ and butylated hydroxytoluene (BHT, BOT)) orig- In accord with previously published Janeros results (lack
inally was examined in rat liver mitochondrial LP system, in of AO for Ca2+ antagonists, nifedipine and nicardipine, even
the presence of Fe2+ ions and using the ultraweak chemilu- at 500 M concentration in LP tests performed on heart
minescence method (Dubur et al. [89]). membrane [134]), no antioxidative activity for 4-phenyl sub-
Several 1,4-DHP derivatives, Hantzsch ester diludine and stituted derivatives of 3,5-dialkoxycarbonyl 1,4-DHP (close
its analogues, were found to be effective antioxidants in this analogues of Ca2+ antagonists) was found, contrary to various
experimental system, changing the kinetics of LP, lengthening 4-nitrophenyl 1,4-DHP derivatives, calcium antagonists, for
the time of the appearance of the maximum of the slow which the significant antioxidant activity was reported [31,
burst of the chemiluminescence (latency, latent period), and 4653].
diminishing the reaction rate (the tangent of the slope angle Studies made on phosphatidylcholine liposomes (our
during the time in which the amplitude of the slow burst unpublished data) suggest approximately three and two
characterizing LP rate increases) and its peak value. Their times more antioxidative activity for 100 M 4-unsubstituted
presence has influenced the reaction constant 6 , in relation DHP compound diludine, when compared to 4-substituted
to a very significant reduction of lipid hydroperoxides and/or DHPs riodipine/nifedipine and nicardipine, respectively, at
inactivation of free radicals, as follows: methemoglobin-induced LP (oxygraphy).
Inhibition of mitochondrial AsA-dependent LP and sta-
ROO + ROO P + h] termination (1)
bilization of mitochondria were shown to be characteristic for
(P = molecular products) or a large group of 1,4-DHP compounds [112], showing to pos-
sess the AOA in simplest in vitro systems (Tirzit and
ROO + ROO + H2 O ROH + RO + 1 O2 (2) Duburs [39], Zilber et al. [44], and Dubur et al. [45])
based on reactions with the stable free radical 1,1-diphenyl-2-
In this study, diludine was one of the most active compounds. picrylhydrazyl (DPPH), LP of fatty acid ester (linethole and
DHPs had activity similar to the standard synthetic AO-BHT methyloleate) emulsions, and phospholipid (phosphatidyl-
(ionol). However, when plotted against applied concentration choline) liposomes. Generally, these properties did not coin-
and time window, diludines activity profile differed from that cide with Ca2+ antagonism. Depending on DHP structure, it
of BHT.
seems that AOA properties are less specific than Ca2+ antago-
There were also similar studies (using different LP rate
nist properties. Both properties may be interrelated but not
experimental detection system and method, Hunter et al.
interdependent.
[133]), based on exploring a group of 26 2,6-dimethyl-
3,5-disubstituted- and 2,6-dimethyl-3,4,5-trisubstituted-1,4- These data show that the presence and the nature of
dihydropyridines (1,4-H2 Py=1,4-DHPs) and five related pyri- a substituent in position 4, as well as 3,5-substituents,
dines as inhibitors of rat liver Mit swelling ( 520 /) and O2 are important factors for 1,4-DHP antioxidant effects in
uptake by ascorbic acid- (AsA-) dependent lipid peroxidation various systems, that is, AsA-dependent nonenzymatic as
and as modulators of Mit swelling induced by Na+ -linoleate well as enzymatic NADPH-dependent lipid peroxidation.
or Na+ -pyrophosphate (Velena et al. [112]). Sometimes, the efficacy of inhibition of nonenzymatic LP
Some of tested 4-DHPs (4-unsubstituted 3,5-dialkoxy- by 1,4-DHPs is higher than the inhibition of the enzymatic
carbonyl-2,6-dimethyl-1,4-DHPs and 3,5-diamido-2,6-dim- LP. However, the action may be opposite, stimulation of the
ethyl-1,4-DHPs, both 4-unsubstituted, or those possessing LP. Hantzsch ester (HEH, diludine) and its close analogues
lipophilic 4-aryl- groups) have shown significant AO and exhibited significant AOA and membrane stabilizing prop-
membrane stabilizing activity. These studies further revealed erties in both AsA-dependent nonenzymatic peroxidation
that 1,4-DHPs preferably act as AO during the stages of of mitochondria and NADPH-dependent enzymatic LP of
initiation and prolongation of LP chain reactions, at low microsomes, usually at similar 10 to 100 M concentrations
concentrations. The studied 1,4-DHPs had IC50 (when 0 / [112].
or /0 = 2) 0.1 M to 100 M and the minimal activity was The order of AO potency (IC50 values) in vitro depends on
scored for oxidized (heteroaromatized) derivatives. drug structure as well as on the experimental conditions and
At the concentration of 100 M, 3,5-di-n-butyloxycarbo- specificity of the biological system. Each method for determi-
nyl-2,6-dimethyl-1,4-DHP entirely stops mitochondrial nation of AOA and ARA has advantages and disadvantages
swelling in the presence of 0.8 mM Na+ -pyrophosphate. (Karadag et al. [135]).
At the same concentration, the following compounds Accordingly, as reported by Gubski et al. [136], IC50 for
alter the mitochondrial swelling rate in the presence of the AsA-dependent LP was 0.25 M and 2.0 M for 1,4-DHP
natural protonophore, Na+ -linoleate: 3,5-di-p-hydroxyphen- Ca2+ antagonists nitrepine (nitrendipine) and nifedipine,
oxycarbonyl- and 3,5-di-p-tolyloxycarbonyl-2,6-dimethyl- respectively. Takei et al.s [137, 138] studies on mitochondrial
1,4-DHPs, 3,5-diethoxycarbonyl-2,6-dimethyl-pyridine (oxi- swelling induced by LP or arachidonic acid in the rat brain
dized form of Hantzsch ester), and more lipophilic 3,5- determined the IC50 values of 12.7, 10.5, 156.8, and 38.4 M for
diamyloxycarbonyl-2,6-dimethyl-pyridine. The alteration of efonidipine, nicardipine, nifedipine, and nimodipine, respec-
swelling may be scored as prolonged, promoted, accelerated, tively. For LDL in the copper-induced oxidation system the
or inhibited. The type of alteration depends on the structure order of potency was vitamin E > felodipine > 2-chlorophenyl
and concentration of 1,4-DHPs, the type of initiators of the analogue of nifedipine > nifedipine > amlodipine, nitrendip-
swelling process, and the medium composition. ine, verapamil, and diltiazem (Rojstaczer and Triggle [119]).
It was interesting to compare the AOA of DHPs with their may be important for their therapeutic effects even in the
susceptibility to oxidation, that is, electron and hydrogen advanced stages of LP.
donating properties.
It has been estimated that electron donor substituents Scheme 2 (stages of initiation, propagation, and termination
in positions 2 and 6 of 1,4-DHP cycle usually promote of lipid peroxidation chain reactions (110)). Initiation and
oxidation, while electron acceptor substituents promote propagation reactions are as follows:
quench oxidation. Stronger electron acceptors in positions 3
(1) HOO + RH R + H2 O2 (RH = membrane lipid)
and 5 also significantly quench oxidation. These estimations
are based on studies including chemical, enzymatic, and HO + RH R + H2 O
electrochemical oxidation of 1,4-DHP derivatives (Dubur and HOO + InH In + H2 O2 (InH = 1,4-DHP)
Uldrikis [70], Duburs et al. [71], and Stradin et al. [139]). HO + InH In + H2 O
On the other hand, diminished AOA of 1,4-DHP relates
to presence of substituents in position 4 (both electron donor (2) R + O2 ROO (R ; RO ; ROO = lipid radicals)
and electron acceptor) (Velena et al. [112]). (3) ROO + RH ROOH + R
3,5-Dicarbamoyl substituents possess minimal quench- (4) ROOH + Fe2+ RO + Fe3+ + HO
ing feature and are followed by benzoyl-, acetyl-, and
alkoxycarbonyl- groups. Maximal decrease was obtained (5) RO + RH ROH + R ; R + InH RH + In
with condensed substituents (i.e., oxoindeno- or oxocyclo- Chain break and termination reactions are as follows:
hexeno- groups) and a CN-group. 4-Unsubstituted 3,5-dicar-
bamoyl derivatives can be easily oxidized and consequen- (6) ROO + ROO P + h] (P = molecular products)
tially inactivated, whereas 4-substituted 3,5-dicarbamoyl-1,4- or ROO + ROO + H2 O ROH + RO + 1 O2
DHPs possess an oxidation potential, analogous to the 4- (7) ROO + InH ROOH + In (ROOH = membrane
unsubstituted 3,5-COOR derivatives. Therefore, they have lipid peroxides)
adequate electron donor properties and are considerably
(8) RO + In Y (Y = molecular products)
stable. This may be the reason for significant membrane stabi-
lization upon exposure to 4-substituted derivatives. Of (9) ROO + Fe2+ Fe3+ + X (X = molecular products)
importance, their AOA was usually more pronounced in (10) RO + RO Y (Y = molecular products)
comparison to 4-unsubstituted derivatives.
Among them, 2,6-dimethyl-3,5-difurfuryloxycarbonyl- In the reversible swelling of mitochondria accompanying
1,4-DHP showed the highest antioxidative activity. In the LP (initiated by mixture of 5 mM GSSG/1 mM GSH), several
group of 3,5-dialkoxycarbonyl derivatives, the strongest 1,4-DHPs showed low or no activity, manifested only as a
activity was attributed to compounds with medium length decrease of the swelling amplitude, without a rate decrease.
alkyl chains (i-butyl-, t-butyl-, and i-amyl- substituents), high An addition of GSH (4 mM) or ATP to swollen mitochondria
level of lipophilicity, minimal electron acceptor properties, caused their contraction in both control and tested system.
and moderate steric hindrance, as contrasted to short or long It may be suggested that 1,4-DHPs, acting as antioxidants
alkyl chain ester derivatives (3,5-dimethoxycarbonyl-, 3,5- in mitochondria, preferably influence LP reactions initiated
diethoxycarbonyl derivatives and 3,5-didodecyloxycarbonyl by ions with variable valency or their complexes with heme
derivative). These data demonstrate the bell-shaped depen- type compounds: methemoglobin, hemin, hematin, and so
dence of AOA on alkyl chain length [112] and are in accord forth (Velena et al. [112]). If the peroxidation process has a
with results obtained in liposomes. However, these data maximal velocity and 50 percent of initial O2 were consumed,
differ from those obtained in emulsions, where diludine was 1,4-DHPs cannot completely break the chain reactions and
the most active compound. Finally, oxidized heteroaromatic prevent subsequent membrane damage: by addition of DHP
derivatives showed only minimal activity. substance at 10 M concentration at the moment of 50
In both LP systems studied (AsA-dependent in mito- percent oxygen consumption, the subsequent oxygen uptake
chondria and NADPH-dependent in microsomes), some of proceeded unchanged. This observation is important for the
1,4-DHPs showed activity similar to classical antioxidant, application of DHPs as inhibitors of initiation and, to a lesser
butylated hydroxytoluene (ionol, BHT) (Velena et al. [112]). degree, propagation stages of LP chain reactions.
However, there was a significant difference related to con- The influence of 1,4-DHPs on Mit swelling is not strictly
centration and incubation time. It allowed us to postu- associated with their own oxidation. There is the possibility
late that 1,4-dihydropyridines (InH), acting as antioxidants- that the labilizing (or stabilizing) effect relates to surface
reductants and scavengers of reactive oxygen species and lipid activity (connected with substituent lipophilicity) or may be
free radicals, preferably influence initiation and propagation the consequence of complexation with some -OH (or -CH3 )
(prolongation) of lipid peroxidation chain reactions (1) group sensitive receptors at the mitochondrial membrane.
(5), according to Scheme 2. The phenomenon is particularly Namely, a bathochromic shift of the absorption band maxi-
prominent in the presence of Fe2+ and other ions of variable mum (about 10 nm) was observed in the visible region before
valency. swelling. However, after swelling in the presence of Na+
Chain break and termination reactions (6)(10) of the linoleate, the spectrum returns to its initial value [112].
LP reaction cascade [89] were influenced by 1,4-DHPs in a Some 1,4-DHPs not only protect mitochondria against
lesser degree than were initiation and propagation steps. This swelling caused by AsA-dependent LP, salts of fatty acids
in vitro [112], but also have beneficial effects on repairing 5-Acetyl(carbamoyl)-6-methylsulfanyl-1,4-DHP-carbo-

their integrity in vivo, after exposure to irradiation, hepato- nitriles (Figure 3) with minor differences in their molecular
toxins, ischemia, hypoxia, or hypothermia. Some of them structure, displaying antioxidant and antiradical activities
were shown to normalize the process of intracellular repara- in vitro, show different biological activities. Namely, 4-p-
tion and physiological regeneration of ultrastructures. They chlorophenyl derivative OSI-1146 displays AO and anti-
were also shown to stimulate reparative processes. If pre- radical activities in cardiovascular OS models, whereas
treated with 1,4-DHPs, irradiated mitochondria will not swell OSI-3701 and OSI-3761 display hepatoprotective activity.
(Ivanov et al. [140, 141]). Thus, these compounds may be potentially useful for treat-
Diludine, ionol, and some other AOs, mitochondria pro- ing several pathological processes, including those associated
tectors, act as anti-ischemic agents. If applied prophylactically with OS (Fernandes et al. [146]). However, besides mito-
in vivo, they may prevent ischemic and reperfusion lesions chondria, the cellular targets for their pharmacological
in heart, kidney, and other organs (Bilenko et al. [113]). The actions have not been fully investigated [146]. All these com-
effect is dependent on applied dose, timing, and way of pounds increase the susceptibility of Mit to MPT. The most
application. When added onto the cryoconservation medium potent is OSI-3701, although it does not affect bioenergetic
for mitochondria preservation, 1,4-DHPs prevented decrease parameters.
of membrane potential, normalized facilitated respiration, Although all these compounds protected mitochondria
and prevented loss of mitochondrial Na+ and Ca2+ ions, against LP induced by the oxidant pair ADP/Fe2+ , OSI-1146
after thawing ([112], see citation number 36 (Subbota et al., was shown to be the most potent. Current data point out
Kharkov, 1984) therein). Diludine was stronger protector, mitochondria as potential targets for protective and toxic
when compared to ionol. actions of DHPs, suggesting that the potential for their use as
CA drug foridone (riodipine) was shown to possess therapeutic agents should also take into consideration their
cardioprotective features, primarily due to is protective effect toxic effects on mitochondria (Fernandes et al. [146]).
on mitochondria exposed to OS [142, 143]. Several structurally different DHP derivatives (antioxi-
Similarly, the DHP water-soluble antiarrhythmic com- dant diludine (diethone), as a 4-unsubstituted DHP, 4-substi-
pound glutapyrone inhibits initiation of LP by free radicals tuted DHPs: CA foridone (bicyclic compound), and the 4-
in erythrocytes and heart mitochondria. Its cardioprotective phenyldiethone compound where phenyl group is joined to
effect has been experimentally shown in heart mitochondrial the DHP in position 4) inhibited the 1-methyl-4-phenyl-
membranes, especially during deep hypothermia (Utno et al. pyridinium iodide (MPP+ ) induced ROS production in
[144]). cerebellar granule cells (CGC) with a distinct potency order:
Cerebrocrast was effective in several translation models foridone (2,6-dimethyl-3,5-dimethoxycarbonyl-4-(o-difluo-
mimicking pathological situations, known to be associated romethoxyphenyl)-1,4-dihydropyridine) > 2,6-dimethyl-3,5-
with cellular OS. The potential protective action of 1,4- diethoxycarbonyl-4-phenyl-1,4-dihydropyridine > diludine.
DHP derivatives (4-substituted compounds: cerebrocrast, They also reversed the MPP+ -induced decrease of the mito-
gammapyrone, glutapyrone, and 4-unsubstituted drug dieth- chondrial membrane potential in the same order (Klimavi-
one) has been studied in rat liver, in experimental models ciusa et al. [147]). Accordingly, it was postulated that the
relevant for oxidative stress and mitochondrial bioenergetics classical two-ring (bicyclic) structure of DHP derivatives
(Fernandes et al. [145]). When succinate was used as the represents an advantage in relation to neuroprotection and
respiratory substrate, higher concentrations (>25 M) of ROS defense and is independent on compounds properties
cerebrocrast depressed respiratory control ratio (RCR), ADP related to calcium ions.
to oxygen ratio (ADP/O), state 3, and uncoupled respiration Novel adamantane-containing 1,4-DHP compounds (Kli-
rates, transmembrane potential (deltapsi), and the phosphate maviciusa et al. [148]) were also found to improve mitochon-
carrier rate. At the same time, state 4 respiration rate was drial functions (MPP+ model) (Klimaviciusa et al. [148]).
three times increased. At concentrations lower than 25 M, Klusa et al. [149] have discovered antineurotoxic effects of
cerebrocrast inhibited mitochondrial IMAC and partially 1,4-DHP taurine derivative, tauropyrone, recorded as Mit
prevented Ca2+ -induced opening of the mitochondrial PTP. function improvement.
Gammapyrone, glutapyrone, and diethone did not induce Many 1,4-DHPs, including Ca2+ antagonists and AO,
these phenomena. When applied at concentrations up to modify LP processes and influence mitochondrial function in
100 M, cerebrocrast, gammapyrone, and glutapyrone did various organs (liver, heart, kidney, and brain) in a different
not affect ADP/Fe2+ -induced LP of mitochondria in rat liver way and degree. Their beneficial action, oxygen or lipid
(as measured by oxygen consumption and TBARS forma- free radical scavenging, antioxidative effects, binding with
tion). On the other hand, low diethone concentrations (up or intercalating into phospholipid bilayer, regulation of ion
to 5 M) inhibited it in a dose-dependent manner. Diethone gating, and regulation of mitochondrial permeability transi-
also prevented against deltapsi dissipation induced by LP tion pores (Tirzit and Duburs [39], Zilber et al. [44], and
initiated by ADP/Fe2+ . Based on these data, it may be Dubur et al. [45]), separately or in combination with each
speculated that cerebrocrast (inhibition of the IMAC) and other, depends on two strong elements: (1) their individ-
diethone (acting as an AO) may provide effective protection ual structure including nature of substituents and their
of mitochondria during OS. Cerebrocrast has shown some positions and (2) the nature of the biological system. For
therapeutic potential for treatment of several pathological example, the direction of LP (inhibition of promotion) was
conditions related to cellular OS [145]. shown to depend on structure and concentration of applied
1,4-DHPs as well as stages of chain reactions. Accordingly, OS associated with hepatic biotransformation of drugs. Var-
mitochondrial swelling may be prolonged (retarded), accel- ious commercial and new nitro-phenyl-DHPs were studied
erated (promoted), or inhibited (Velena et al. [112]). against LP using rat liver microsomes under oxidative stress
Therefore, there is a ground for 1,4-dihydropyridines, [159].
either Ca2+ antagonists or antioxidants, to be nominated as Incubation of rat liver microsomes with the 4 -nitro-
useful tools in development of mitochondrial drugs related 4-phenyl-1,4-DHP compounds (2,6-dimethyl-4-(4 -nitro-
to the control of OS. phenyl)-1,4-dihydropyridin-3,5-diethyl-dicarboxylate and
N-ethyl-2,6-dimethyl-4-(4 -nitrophenyl)-1,4-dihydropyri-
(c) DHPs as AOs in Endoplasmic Reticulum (Inhibition of din-3,5-dimethyl-dicarboxylate) results in an inhibition
NADPH-Dependent LP System): Inhibition of NADPH Oxi- of LP, the UDPGT (UDP-glucuronyltransferase) oxidative
dase by DHPs. Elevated level of NADPH oxidase 4- (NOX4-) activation, and the microsomal thiol oxidation induced by
derived hydrogen peroxide (H2 O2 ) joined with concomitant Fe3+ /ascorbate, a generator system of ROS. This effect was
decrease of nitric oxide (NO) mediated signaling and reac- also produced by nitrofurantoin and naphthalene in the
tive oxygen species scavengers are considered to be central presence of NADPH.
factor in molecular pathogenesis of fibrosis (Sampson et Interestingly, IC50 of DHPs obtained from microsomal LP
al. [150]). Inhibition of microsomal NADPH-dependent LP, assays decreased to the same extent as the microsomal thiols
with particular focus on NADPH oxidases (NOX15 and
oxidation provoked by Fe3+ /ascorbate [159]. Nevertheless,
DUOX1), may be very important for neuro-, cardio-, and
the AO effects of a nitrophenyl-DHP compound, in which
hepatoprotection (Velena et al. [112], Leto and Geiszt [151],
hydrogen at position one of the DHP ring was replaced by
Griendling et al. [152], and Chen et al. [153]). Endoplasmic
the ethyl group, were significantly weaker. Authors speculated
reticulum may be an important target, as this is where 1,4-
that DHPs can resemble NADH, transferring one hydrogen
DHPs could display their antioxidative properties (Velena et
atom of 4-position (H ) to anion superoxide and another of
al. [112], Leto and Geiszt [151], Griendling et al. [152], and
the 1-position (H+ ) by way of a cationic radical intermediate
Chen et al. [153]).
to generate pyridine derivatives and water [159].
However, the initiation of LP in the NADPH-dependent
microsomal system does not appear to involve either super- The AO effects of various tested DHP derivatives (m- and
oxide or hydrogen peroxide, since neither SOD nor cata- p-NO2 phenyl as well as methyl or ethyl and isopropyl-DHP
lase can inhibit it. On the other hand, reduced iron plays 3,5-dicarboxylate derivatives) were not significantly different.
an important role in both the initiation and propaga- The authors assumed that the -NH- group of the dihydropy-
tion of NADPH-dependent microsomal lipid peroxidation ridine ring could contribute both to the development of
(Hochstein and Ernster [154] and Repetto et al. [111]). the calcium channel antagonism and to the antioxidative
Many DHPs possess inhibitory activity not only towards properties of DHPs [159].
AsA-dependent LP in mitochondria but also towards Prevention of the membrane LP seemingly depends on
NADPH-dependent LP, as shown in isolated rat liver micro- the concentration of potential antioxidants, such as vitamin
somes (Velena et al. [112]). This means that these compounds E or even 1,4-DHP in lipids. However, only the differences
interact with the shared parts (nonenzymatic and enzymatic) in synthetic DHPs lipophilicity cannot explain significant
of LP pathways. variations of DHPs concentration in microsomal membrane
Microcalorimetry and fluorescent probes procedures and cannot clarify the strength of their antioxidative activity.
were used for studying the interaction of alpha-tocopherol This work [159] has further demonstrated that 1,4-DHPs
and 1,4-DHPs with endoplasmic reticulum membranes and may prevent the OS induced by biotransformation of some
model systems, human serum albumin, and phospholipid drugs, for example, antibiotic nitrofurantoin. Simultaneous
bilayers [155]. Modification of microviscosity of the endo- administration of DHPs and nitrofurantoin may be beneficial
plasmatic reticular membranes depends on localization of in reducing nitrofurantoin side effects.
antioxidants within the protein structures or phospholipid While most of Ca antagonist 1,4-DHPs are metabolized
phase. Increase of membrane structuralization under the by CYP3A4 (Guengerich et al. [160]), not all of them are good
influence of 1,4-DHPs blocked their antioxidant action in inhibitors of its activity. Thus, nicardipine, but not nifedipine
spontaneous and induced lipid peroxidation. and nitrendipine, inhibits CYP3A4 in vitro [53]. Interaction
Inhibition of rat heart and liver microsomal lipid perox- of different DHPs with various types of cytochrome P450 was
idation by nifedipine was observed [156], while Goncalves et described by Carosati et al. [53]. It was also reported that
al. [157] found antioxidant effect of calcium antagonists on DHP class calcium channel blockers reduce the antiplatelet
microsomal membranes isolated from different brain areas. effect of clopidogrel (Park et al. [161]). This implies the mutual
Nitroaryl-1,4-DHPs are both calcium channel antagonists interactions of both drugs with CYP3A4.
and antioxidant agents (Letelier et al. [158, 159]), commonly
used for treatment of cardiovascular diseases. These drugs (3) In Vivo. Evaluation of nifedipine effects on Saccharomyces
must be metabolized through cytochrome P450 oxidative cerevisiae was recently published (Asma and Reda [162]). Sur-
system (NADPH-cytochrome P450 reductase), mainly local- prisingly, nifedipine exercised a toxic effect on Saccharomyces
ized in the hepatic endoplasmic reticulum. Several lipophilic cerevisiae shown through measuring cellular proliferation,
drugs generate OS while being metabolized by this cellular respiratory activity, and the level of some biomarkers (CAT
system. Thus, DHP antioxidant properties may prevent the and MDA).
However, majority of data obtained on various animal achieving and maintaining an adequate concentration may be
cells and tissues by other authors show the protective role of problematic both in vitro and in vivo.
DHPs against both LP and oxidative stress [113, 163, 164].
The AOA attributed to many 1,4-DHPs, Ca2+ antag- (b) Role of Concentration and Lipophilicity (Membrane/Water
onists and other compounds, reflecting on catalytic LDL or Lipid/Water Partition Coefficients) of DHPs in Their Action
peroxidation (see Section 3.3.1 (2) and Section 3.5), should as AOs and Antiradical Compounds. Antioxidative effects of
encourage their testing for treating cardiovascular diseases any antioxidant depend on its concentration at the site of
and/or alterations of lipid metabolism. action. This parameter is hardly measurable, especially in
The possibility that 1,4-DHP-based calcium antagonists two-phase systems, representing one of obstacles in com-
exert an antiatherosclerotic action (via inhibition of LDL parison to AOA upon applying various compounds [172].
oxidation and other mechanisms) has been proved by many It is often incorrectly assumed that the concentrations in
experimental data [165] and several clinical trials. Besides the aqueous solution and at the site of action are the same.
antihypertensive effect, nicardipine was shown to possess However, even when the concentration in the aqueous phase
antioxidative and antielastase activity [165, 166]. These prop- may be well controlled, the concentration at the site of
erties may be useful for prevention of inflammatory reaction action in the lipid matrix of the membranes might fluctuate
which is relevant for hypertension pathogenesis. between different test compounds, depending on a difference
1,4-DHPs administration inhibits LDL oxidation medi- in lipophilicity [175]. The prevention of the membrane LP
ated by oxygen radicals, leading to decreased carotid inti- also seems to be dependent on the DHP concentration in the
mal media thickness and reduced progression of coronary lipid matrix (Mason and Trumbore [46]) and its amphiphilic-
atherosclerosis [130]. It additionally preserves Apo B-100 ity. For example, AOA of diludine is associated with its
integrity against ROS. Of importance, antiatherogenic mech- lipophilicity and consequential ability to be incorporated
anisms differ between animals and humans (primarily in into liposomes (Panasenko et al. [176]). It was also found
the stage of conversion of aldehydes to carboxylic acids) that diludine easily incorporates into the outer monolayer of
(Parthasarathy et al. [167]). erythrocyte membranes [176].
For example, furyl-DHP compound (FDP-1, diethyl 2,6- Membrane/buffer partition coefficients (lambda) were
dimethyl-4-(furyl)-1,4-dihydropyridine-3,5-dicarboxylate) directly measured in the sarcolemma and sarcoplasmic
was shown to act as an antioxidant (decreasing MDA, GOT, reticulum membranes for three CA DHPs. The obtained
and FFA release of ischemic myocardium and inhibiting Ca- values were in a broad range between 5000 and 150000
ATPase of erythrocyte membranes), preventing against heart (Herbette et al. [177]). These drugs interact primarily with the
myocardium ischemia-reperfusion injury and arrhythmia, membrane bilayer component but may also bind to proteins,
when applied (in rats) at 10 mg/kg (Liu et al. [168]). both nonreceptors and receptors. The intrinsic forward rate
Similarly, antioxidative effects of azelnidipine and constants for DHP binding to sarcolemmal calcium channel
amlodipine prevented neuronal damage by CCBs, after receptors were apparently not strongly dependent on their
transient focal ischemia in rats (Lukic-Panin et al. [169]). membrane partition coefficients. For example, nimodipine
Allanore et al. [170] found that both nifedipine and (lambda = 6300) had a forward rate constant of 6.8 0.6
nicardipine significantly decrease the mean level of plasma 106 /M/s, whereas the forward rate constant for Bay P 8857
markers for oxidative stress in patients suffering from sys- (lambda = 149000) was 1.4 0.8 107 /M/s. Since these DHPs
temic sclerosis. are highly liposoluble, model calculations for this binding
Antioxidants may be considered as promising neuropro- reaction demonstrated that these rates on lipid solubility
tective compounds. Still, while experimental data demon- would probably not be reflected in the experimental forward
strate neuroprotective effect in vitro and in animal mod- rate constants. In addition, the intrinsic forward rate constant
els, clinical evidence is still unsatisfactory and insufficient for nimodipine binding to sarcolemmal calcium channel
[171]. receptors was found not to be linearly dependent on the
viscosity of the buffer medium over a fivefold range. The rate
(a) Role of Metabolism of DHPs in Their AOA. Metabolic of drug nonspecific binding to nonreceptor protein present in
pathways and bioavailability of the probable AOA com- highly purified sarcoplasmic reticulum membranes appears
pound determine antioxidant activity in vivo. Antioxidant to be extremely fast, at least 103 times faster than specific
metabolites may vary in stability and activity leading to two drug binding to the receptor in the sarcolemma. Authors
opposite scenarios: lack or presence of activity, substantially concluded that partitioning into the lipid bilayer matrix of
contributing to the overall AOA [172]. Metabolic biotransfor- the sarcolemma could be a general property of CA DHPs
mation of DHPs includes oxidation (heteroaromatization), and may be a prerequisite for their binding to sarcolemmal
side chain ester group cleavage (deesterification), and 4- membrane receptors (Herbette et al. [177]).
substituent abstraction a.o. [160]. None of the DHPs metabo- The binding of DHP calcium channel agonists and antag-
lites was shown to be more toxic than original, reduced form onists (including those with AO properties) to receptors in
of the compound. The commonly detected metabolites of cardiac sarcolemmal membranes is a complex reaction that
the DHPs do not seem to possess the AO activity (with may involve an interaction with the lipid bilayer matrix of
some exceptions as in the case of metabolites of nifedipine the sarcolemma (Herbette et al. [178]). Belevitch et al. [179]
and its analogues, including nitrosonifedipine [173, 174]) (see studied the binding of DHP CCBs (riodipine and nifedip-
further in Section 3.5). Due to DHPs intrinsic instability, ine) and verapamil to model and biological membranes by
fluorescence analysis. The consistent location of Ca agonist Bay K 8644 was significantly more effective in scavenging
Bay K 8644 was determined to be within the region of superoxide anions than lacidipine, Bay O 5572, or nifedipine
the first few methylene segments of the fatty acyl chains of (micro- to millimolar concentration range). Addition of an
the membranes (Mason et al. [180]). This position is near artificial membrane preparation resulted in an enhanced
to that observed for the DHP calcium channel antagonists AO effect, with lacidipine being the most effective DHP in
nimodipine and Bay P 8857. quenching radicals (low micromolar concentration range).
The majority of studies on OS were performed with DHPs In the third model, mimicking hyperglycemia (30 mmol/L),
with various lipophilicity, but only a few studies reported nifedipine was significantly more potent antioxidant (ther-
amphiphilicity of DHP derivatives. Amphiphilic DHP deriva- apeutical nanomolar concentration range) than the other
tive K-2-11 reduced the cellular generation of ROS. It also DHPs. Calculated lipophilicity of these four substances
revealed complete reversal of multidrug resistance (MDR) of (lacidipine > Bay O 5572 > Bay K 8644 > nifedipine) was
the resistant cells. K-2-11 was more efficient than well-known positively correlated with antioxidative potential only in the
MDR inhibitor verapamil. Cytotoxic effects of anticancer second experimental model. It has been concluded that AO
drug doxorubicin were enhanced by K-2-11 in both MDR and properties of DHP substances need to be tested in various
parental, nonresistant cell line (Cindric et al. [181]). K-2-11 models for demonstrating that nifedipine exhibits ROS-
suppresses increase of ROS and consequentially prevents NF- quenching properties in a therapeutic concentration range
B activation leading to decreased expression of MDR1 and [184].
increased expression of antiapoptotic genes. This signaling
switch is necessary for restoring the chemosensitivity of 3.4.1. AOA of DHPs in Isolated Cells and Cell Cultures
cancer cells. This phenomenon is characteristic both for (Comparison with Other Simplest Systems). Although DHPs
1,4-DHPs [182] (18 novel asymmetrical DHPs bearing 3- possess neuromodulatory and/or antimutagenic properties,
pyridyl methyl carboxylate and alkyl carboxylate moieties the mechanisms of action related to these phenomena are
at C3 and C5 positions, resp., as well as nitrophenyl or not entirely elucidated. Borovic et al. [185] have studied 1,4-
heteroaromatic rings at C4) and for their oxidized forms, dihydroisonicotinic acid (1,4-DHINA) derivatives of 1,4-
pyridine compounds (Zhou et al. [183]). DHP, water-soluble analogues of a well-known AO diludine
(diethone): 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydro-
3.4. Dependence of AOA of DHPs on the Experimental System. isonicotinic acid, sodium 2-(2,6-dimethyl-3,5-diethoxycar-
AO effect of DHPs depends on their structure and the bonyl-1,4-dihydropyridine-4-carboxamido)glutamate, gluta-
experimental system used (in vitro model system, subcellular pyrone and sodium 2-(2,6-dimethyl-3,5-diethoxycarbonyl-
organelle, and cells, ex vivo and in vivo). Ideally, for the 1,4-dihydropyridine-4-carboxamido)ethane-sulphate, tauro-
evaluation of the profile and value of DHPs AO properties, pyrone as AO and bioprotectors (Figure 4).
each compound should be tested in as many systems as 1,4-DHINAs activities were studied in comparison to
possible (Dubur et al. [45]). Trolox by N,N-diphenyl-N -picrylhydrazyl (DPPH ), deoxy-
Lipidomics studies have been traditionally explored for ribose degradation, ABTS radical scavenging, and AOA
studying AOA of DHPs. Proteomics methods are less rep- (antioxidative capacity method) assays; copper-induced LP
resented and are mostly focused on the properties of DHPs of cultured rat liver cells (MDA determination by HPLC
related to scavenging of protein free radicals. So far, there are and 4-hydroxynonenal-protein conjugates by dot-blot); 3 H-
no studies on the role of DHPs in scavenging nitrosoperoxy- thymidine incorporation and trypan blue assay for liver cells
carbonate, the reactive species formed out of peroxynitrite, in growth and viability. Ic decreased the amount of 4-HNE-
the presence of carbon dioxide. Although it was shown that protein adducts. In all assays, Ia was the most potent AO,
albumin binds diludine, no studies revealed the relevance of able to completely abolish copper induced LP of liver cells,
this effect for the AOA of diludine. while Ic only slightly decreased it. Thus, AOA is important
There are findings showing that dihydropyridine calcium activity principle of Ia, which was even superior to Trolox in
antagonists (DHPs CA) could indirectly play a beneficial, treated cell cultures. Ia (and its analogues) are easily oxidized
protective role during development of atherosclerosis. in the Fenton system (Rubene et al. [186]), exerting ARA too.
Namely, Berkels et al. [184] have studied antioxidative
properties of four substances: the DHP prototype CA, 3.5. Peculiarities Related to Antioxidative and Antiradical
nifedipine, the long-acting CA, lacidipine, the DHP calcium Activity of Some 1,4-DHPs: Ca Antagonists. Nine commer-
channel agonist, Bay K 8644, and the bulky DHP derivate, Bay cialized, structurally and functionally different DHPs, CA,
O 5572, in three different models: (1) in an in vitro superoxide will be discussed further. Their common feature is ability to
anion generating system (hypoxanthine/xanthine oxidase) prevent OS. This also counts for some of their metabolites,
for testing the pure antioxidative effect, (2) in an artificial as already discussed. The comparative effects of some DHPs,
membrane preparation (dimyristoylphosphatidylcholine) for CA, on oxidative stress-induced modification of LDL were
mimicking a more physiological environment, and (3) under already reviewed in Section 3.3.1 (2)-(a). AOA of CA DHPs
conditions of stimulated ROS release (hyperglycemia) from was discussed in Sections 3.3.1 (2)-(b) and 3.3.1 (2)-(c).
native endothelial cells derived from porcine coronary arter-
ies. 3.5.1. Nifedipine and Its Close Analogues. Nifedipine, vera-
The study also revealed the potential correlation between pamil, and antiarrhythmic-antihypoxic drug, stobadin, were
lipophilic and AO properties of DHPs. In the first model, shown to depress lipid peroxidation of phosphatidylcholine
COONa
COOH CONHCH(CH2 )2 COONa CONH(CH 2 )3 COONa
C2 H5 OOC COOC2 H5 C2 H5 OOC COOC2 H5 C2 H5 OOC COOC2 H5

H H H
1,4-DHINA Glutapyrone Gammapyrone
CONH(CH2 )2 SO3 Na
C 2H 5OOC COOC 2H 5
H3 C N CH3
H
Tauropyrone
Figure 4: Derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA).
liposomes (Ondrias et al. [187]). However, data obtained in (1) Metabolites of Nifedipine and Its Analogues as Antioxidants
some other experimental systems are conflicting. and Regulators of OS. Antioxidant activity of nifedipine,
In an in vitro model of sarcolemmal membrane lipid per- 3,5-dimethoxycarbonyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-
oxidation, three calcium blockers (nifedipine, verapamil, and dihydropyridine, was originally studied in vitro by Kirule et
diltiazem) exhibited concentration-dependent (10400 M) al. [195] and Tirzit et al. [196]. According to the kinetic data
inhibitory effects [188, 189]. Nifedipine, the most effective of peroxide accumulation and the ESR spectra (inhibition of
calcium blocker, was more than two-fold potent compared to the autoxidation of methyl oleate in presence of nifedipine)
propranolol, achieving significant effect at 10 M. Nifedipine AO action was exerted by the formation of nitroso analogue
protective role on LP using reduced glutathione as model of the oxidized nifedipine, nitroso nifedipine: 2,6-dimethyl-
marker was recently described (Ray et al. [190]). Antiper- 4-(2-nitrosophenyl)-3,5-pyridine dicarboxylate (NO-NIF).
oxidative properties of CA nifedipine and its analogues This nitroso aromatic derivative can form nitroxyl radicals
were explored in different systems/pathogenic processes: exhibiting remarkable AOA in the presence of unsaturated
atherogenesis (Henry [165]), brain focal ischemia (Yamato fatty acids and lipids [196].
et al. [191]), nephroprotection related to cyclosporine intake The primary species of free radicals that have been
(Chander and Chopra [192]), and hepatoprotection related obtained and identified were ion radicals of the nitrophenyl
to intake of diethyldithiocarbamate (Gaafa et al. [193]). type (Ogle et al. [76]). Such a mechanism coincides with
Recent data suggest that nifedipine action as protector for mechanisms proposed afterwards by Nunez-Vergara et al.
endothelial cells proceeds independently from its CA proper- [96], Lopez-Alarcon et al. [103], Valenzuela et al. [104],
ties. Fukuhara et al. [174], and Yanez et al. [197].
The absence of antioxidant effects of nifedipine and There are also data showing that nitroso compounds may
diltiazem on myocardial membrane lipid peroxidation, oppo- inhibit LP by direct radical trapping and subsequent forma-
site to nisoldipine and propranolol, was also described tion of stable nitroxide radicals. It was further found that the
[194]. Nisoldipine and propranolol were shown to have reactivity between the synthesized 1,4-DHP derivatives with
a concentration-dependent antiperoxidant effect, with IC50 alkylperoxyl radicals involves electron transfer reactions. This
values of 28.2 and 50.1 M, respectively. Finally, nisol- is documented by the presence of pyridine as a final product
dipine appeared to possess dual antiperoxidant mecha- of the reaction and complete oxidation of the nitroso group
nisms, involving both preventive and chain-breaking proper- in the case of the nitrosoaryl 1,4-dihydropyridine derivatives
ties. (Valenzuela et al. [104]). Tested compounds reacted faster
These findings were confirmed in some other studies, toward alkylperoxyl radicals and ABTS radical cation than
including reports on the lack of antioxidative activity of alkyl ones (Lopez-Alarcon et al. [103]).
nifedipine and nicardipine, even at 500 M concentration Nitrosonifedipine, a photodegradation product of nifedi-
in heart membrane lipid peroxidation tests [134]. Similarly, pine, significantly recovers cellular damage induced by tumor
ROS formation in bovine aorta smooth muscle cells was not necrosis factor-alpha. It also prevents toxic effects of cumene
affected by addition of amlodipine, nimodipine, and nife- peroxide which hampers integrity of cell membranes through
dipine [123]. oxidative stress. Its positive effects are equal to Trolox-C.
As a result, nitrosonifedipine was already a long time ago 3.5.2. Lacidipine. It is a generic DHP type antihypertensive
claimed as a candidate for a new class of antioxidative drugs CA, 3,5-diethyl 4-{2-[(1E)-3-(tert-butoxy)-3-oxoprop-1-en-1-
(Kirule et al. [195]), cellular protectors against oxidative stress yl]phenyl}-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxy-
in glomerular endothelial cells [174]. late.
Moreover, Misik et al. [198], Ondrias et al. [199], and Ursini [204] described redox behaviour of lacidipine and
Stasko et al. [200] studied AOA of nifedipine and its oxidized showed its tissue protective features. Cristofori et al. studied
nitroso analogue. NO-NIF prevents the progression of type antiatherosclerotic activity, in addition to lacidipines CA
2 diabetic nephropathy associated with endothelial dysfunc- and AO properties [205]. Lacidipine reduced the extent of
tion through selective AO effects (Ishizawa et al. [201]). NO- atherosclerotic area in hypercholesterolemic apoE-deficient
NIF administration reduces albuminuria and proteinuria mice (these mice show widespread vascular lesions which
as well as glomerular expansion without affecting glucose closely resemble the inflammatory fibrous plaques seen in
metabolism or systolic blood pressure. NO-NIF also sup- humans in atherosclerosis). The reduction may be associated
presses renal and systemic OS and decreases the expression with the capacity of the drug to maintain endothelial NO
of intercellular adhesion molecule-1 (ICAM-1), a marker of levels at concentrations useful to protect against vascular
endothelial cell injury, in the glomeruli of the KKAy mice. damage. This work suggested that DHPs modulate vascular
Similar effects were achieved in endothelial nitric oxide syn- relaxation via increased release of NO.
thase (eNOS) knockout mice. Moreover, NO-NIF suppresses Herbette et al. [178] remarked optimal hydrophobicity of
urinary angiotensinogen (AGT) excretion and intrarenal lacidipine due to cinnamic acid substituent, so membrane
AGT protein expression in proximal tubular cells in the interactions and facilitation of the treatment of atherosclero-
KKAy mice. On the other hand, hyperglycemia-induced sis could proceed (see also Section 3.3.1 (2)-(a)).
mitochondrial superoxide production was not attenuated by
NO-NIF in cultured endothelial cells. 3.5.3. Amlodipine. Amlodipine (Norvasc), (RS)-3-ethyl
Fujii and Berliner found EPR evidence for free radical 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-
adducts of nifedipine in vivo [202]. The nature of these rad- 6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (AML), has
icals was surmised by comparing the reaction of illuminated an antioxidant effect on vessels in vitro and is a 3rd genera-
nitrosonifedipine with polyunsaturated fatty acids. Surpris- tion of charged dihydropyridine CCB that is widely used for
ingly, identical radical spectra were detected from excised the treatment of hypertensive patients.
liver doped with nonilluminated nifedipine, suggesting that Amlodipine was shown to have the highest affinity
(amlodipine > verapamil diltiazem) for the membrane
this drug can be enzymatically converted in vivo to its nitroso
bilayer ( = 104 ). It produced the significant changes in
analogue without the requirement for illumination. This is
membrane thermodynamic properties, including a reduction
one of the first reports of in vivo EPR evidence for a class of
in the thermal phase transition temperature (11%), enthalpy
unsaturated fatty acid radical conjugates resulting from the (14%), and cooperative unit size (59%), relative to the
normal metabolism of a common drug. control, phosphatidylcholine liposomes (Mason et al. [49]).
Daz-Araya et al. [173] studied some 4-nitrophenyl-DHPs Amlodipine AOA is related to its reductant nature or
on Fe3+ initiated LP in rat brain slices. LP, as measured hydrogen donor properties, respectively. Its ability for donat-
by MDA formation, was inhibited by all the tested nitro- ing protons and electrons to the lipid peroxide molecules
aryl derivatives of 1,4-DHP over a wide range of concen- blocks the LP process.
trations. On the basis of both time course and IC50 experi- Amlodipine and even its enantiomers (Zhang et al. [206])
ments the tentative order of AOA on rat brain slices was act as ROS and NOS effectors in several model systems of OS.
nicardipine > nisoldipine > (R,S/S,R)-furnidipine > (R,R/ Antioxidant properties of amlodipine were recently reviewed
S,S)-furnidipine > nitrendipine > nimodipine > nifedipine. by Vitolina et al. [32]. Both in vitro and in vivo studies
1,4-DHP derivatives that lack a nitro group in the mole- of amlodipine AO properties revealed inhibition of lipids
cule (isradipine and amlodipine) also inhibited LP in rat oxidative damage, primarily those associated with cellular
brain slices but at higher concentrations than that of nitro- membranes and lipoprotein particles (LDL) (Mason et al.
substituted derivatives. All tested compounds reduced and [50]).
oxidized nitrosoaryl derivatives (2,6-dimethyl-4-(2-nitro- Under controlled experimental conditions in vitro amlo-
sophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (pho- dipine showed AOA and ARA, by inhibition of lipid peroxide
formation and trapping ROS. Its scavenging activity for
tooxidation product of nifedipine NTP) a.o.) and were
hydroxyl and peroxyl radicals at concentrations as low as
more potent inhibitors of LP than their parent molecules
10.0 nmol/L (which is remarkably less compared to the classi-
(Valenzuela et al. [104]).
cal antioxidants, GSH, uric acid, and Trolox) was shown to be
The electrooxidation process of 4-nitrosoaromatic DHPs independent of the calcium channel modulation (Franzoni et
is a strongly pH-dependent (two-electron two-proton mech- al. [207]).
anism): ECEC type of mechanism, that is, the sequence: AML showed efficiency as scavenger of peroxyl radicals
e /H+ /e /H+ at pH > 8.5; ECCE mechanism (e /H+ /H+ /e ) (TOSC assay: 5945 544 units/mg), significantly stronger
at pH < 8.5 dominates. Reduction reaction of nitroso group (>50%, < 0.001) than GSH (2733 636 units/mg) and 70%
is as follows: R-NO + 2e + 2H+ RNHOH (Bollo et al. weaker ( < 0.0001) than uric acid (18144 696 units/mg)
[203]). and Trolox (17522 734 units/mg).
Of interest, the scavenging capacity of AML towards (in both doses). Nimodipine cannot induce these effects via
hydroxyl radicals (1455 154 units/mg) was 320% higher blockade of Ca2+ channel.
( < 0.00001) than that of GSH (358 112 units/mg), 20% Ismailoglu et al. [213] studied the therapeutic effects
higher than that of uric acid (1198 121 units/mg), and 100% of melatonin and nimodipine in rats after cerebral cortical
higher than that of Trolox (759 143 units/mg). injury. These beneficial effects in rats after cerebral cortical
Amlodipine was shown to increase enzyme activity of injury seemed to be related to AOA of nimodipine.
paraoxonase (PON) and glutathione peroxidase (GSH-Px).
However, it also decreases glutathione reductase (GSSG-R) 3.5.6. Benidipine. Licensed in Japan and South Asia as CA
activity and diminishes the concentration of the endogenous (CCB) benidipine possesses AO properties. Chemically, it is
antioxidant -tocopherol (vitamin E). Moreover, AML in a 5-methyl 3-[(3R)-1-(phenylmethyl)piperidin-3-yl] 2,6-dim-
concentration of 2 ng/mL decreased the content of malonic ethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxy-
dialdehyde and activity of superoxide dismutase in the blood late (or its hydrochloride, (4R)-rel-3,5-pyridinedicarboxylic
(Gatsura [208]). acid, 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-, 3-methyl
Verapamil and amlodipine produced a potent anti- 5-[(3R)-1-(phenylmethyl)-3-piperidinyl] ester, hydrochloride
ischemic effect and reduced area of myocardial infarction in (1 : 1)).
rats. The observed changes were accompanied by inhibition Benidipine influences processes connected with OS in
of LP. In contrast to verapamil, in vitro application of AML in several ways. It prevents lysophosphatidylcholine- (lysoPC)-
a dose of 50 ng/mL decreased hemoglobin affinity for oxygen. induced injury and ROS production in human aortic
When present in a concentration of 2 ng/mL, AMD decreased endothelial cells (HAECs) (Matsubara and Hasegawa [214]).
the content of MDA and activity of SOD in the blood. Matsubara et al. [215] explained this effect, based on stimula-
On the other hand, amlodipine shows no activity related tion of nitric oxide release.
to inhibition of macrophage superoxide release and cell LysoPC is a component of oxidized low-density lipopro-
migration, which occurs as a consequence of decreased TNF teins (oxLDLs), which plays an important role in the patho-
induced O2 release. genesis of atherosclerosis. Pretreatment with benidipine (0.3
Amlodipine-induced reduction of OS in the brain is 3 mol/L) for 24 h protected against lysoPC-induced cyto-
associated with sympathoinhibitory effects in stroke-prone toxicity in the HAECs through inhibition of both lysoPC-
spontaneously hypertensive rats (SHRSP) (Hirooka et al. stimulated ROS production and caspase-3/7-like activation,
[209]). Antihypertensive treatment with amlodipine reduced with a similar potency. Since caspase-3/7 is involved in
OS in all examined areas of the brain and decreased blood executing the apoptotic process, the reduction of the activity
pressure without a reflex increase in sympathetic nerve of this enzyme by benidipine may explain the antiapoptotic
activity. Nicardipine, another CA DHP, surprisingly, was effect of the drug. However, benidipine did not suppress
significantly less active than amlodipine. lysoPC-induced phosphorylation of mitogen-activated pro-
tein kinases and Ca2+ influx in HAECs. These results suggest
that the antioxidant properties of benidipine may be respon-
3.5.4. Lercanidipine. Tomlinson and Benzie reported AO sible for its ability to inhibit ROS production, a possible
effect of lercanidipine [210], which is well known as anti- reason for reduced activation of caspase-3/7. In conclusion,
hypertensive drug Zanidip, 2[(3,3-diphenylpropyl)(methyl)- benidipine suppresses lysoPC-induced endothelial dysfunc-
amino]-1,1-dimethylethyl methyl 2,6-dimethyl-4-(3-nitro- tion through inhibition of ROS production, which is due at
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate. Compara- least in part to its antioxidant effect, and not through the
tive data about this drug AOA were presented in parts of this inhibition of L-type voltage-dependent calcium channels.
paper about other individual CA DHPs and in the part about Matsubara and Hasegawa [216] examined the effects
the ex vivo DHPs effects on LDL. of benidipine on cytokine-induced expression of adhesion
molecules and chemokines (chemoattractants), which are
3.5.5. Nimodipine. Nimodipine (ND), commercially known important for the adhesion of monocytes to endothelium.
as Nimotop, is 3-(2-methoxyethyl) 5-propan-2-yl 2,6-dim- Pretreatment of HAECs with benidipine (0.310 mol/L)
ethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxy- for 24 h significantly suppressed cytokine-induced vascular
late. It is centrally active CA. cell adhesion molecule-1 (VCAM-1) and intracellular cell
Treatment with glutathione blocked and with nimodipine adhesion molecule-1 (ICAM-1) mRNA and protein expres-
attenuated neuronal cell death, caused by prolonged exposure sion, resulting in reduced adhesion of THP-1 monocytes.
of cell culture to 4-HNE (Faraqui [211]). Benidipine also suppressed induction of monocyte chemoat-
Nascimento et al. [212] found AO effect of nimodipine in tractant protein-1 (MCP-1) and interleukin-8. Benidipine
young rats after pilocarpine- (PIL-) induced (in 400 mg/kg) also inhibited redox-sensitive transcriptional nuclear factor-
seizures. The PIL administration increased the striatal cata- B (NF-B) pathway, as determined by Western blotting of
lase (CAT) activity. The administration of ND, 30 mg/kg, inhibitory B (IB) phosphorylation and luciferase reporter
30 min before PIL, preserved normal value of CAT activity. assay. Results of analysis using optical isomers of benidip-
On the other hand, no difference was detected in the animals ine and antioxidants suggest that these inhibitory effects
treated with lower dose, 10 mg/kg. These results confirm were dependent on pharmacological effects other than Ca2+
the neuroprotective/antiepileptic effect of ND in young rats, antagonism. Benidipine may thus have anti-inflammatory
suggesting that this drug acts positively on lipid peroxidation properties and benefits for the treatment of atherosclerosis.
Benidipine was also shown to inhibit ROS production in and microalbuminuria, when compared to patients treated
polymorphonuclear leukocytes and oxidative stress in salt- with the combination (2). Because diabetes is associated with
loaded stroke-prone spontaneously hypertensive rats (Mat- severe chronic OS the observed results might be at least in a
subara et al. [217]). part due to the AOA of azelnidipine.
It should be mentioned that other DHPs also have In favor of this are findings of Abe et al. [224] who found
endothelial AO actions [218]. additive antioxidative effects of azelnidipine on angiotensin
receptor blocker olmesartan treatment for type 2 diabetic
patients with albuminuria.
3.5.7. Azelnidipine (AZL). Azelnidipine, 3-[1-[di(phen- Similarly, the AOA of thiazolidinediones (insulin sensi-
yl)methyl]azetidin-3-yl] 5-propan-2-yl 2-amino-6-methyl- tizer) and their effect on cardiovascular function in type 2
4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate diabetic model rats and also those of some DHPs (nifedipine,
(AZL), CAS number: 123524-52-7, is commercially available amlodipine, or AZL, commonly used antianginal and anti-
4-nitroaryl-DHP type calcium antagonist with long-acting hypertensive agents) in cultured human endothelial cells LP
antihypertensive action (long-acting CA (CCB)) and a low were examined (Mizushige [225]). The AOA was evaluated
reported incidence of tachycardia. It additionally possesses by measuring 8-iso-prostaglandine F2 concentration and
beneficial effects in OS and diabetic conditions. azelnidipine exhibited potent AOA.
Azelnidipine prevents cardiac dysfunction in strepto- Insulin (INS) resistance combined with hyperinsuline-
zotocin-diabetic rats by reducing intracellular calcium accu- mia is involved in the generation of OS. A relation-
mulation (altering intracellular Ca2+ handling proteins), OS, ship exists between increased production of ROS and the
and apoptosis (Kain et al. [219]). AZL can reduce the superox- diverse pathogenic mechanisms involved in diabetic vascular
ide production. It exerts its protective effects by targeting the complications, including nephropathy. Manabe et al. [226]
NADPH oxidase and mitochondrial redox enzymes. AZL- revealed that high doses of INS augmented mesangial cell
treated diabetic rats express enhanced level of bcl-2 in the proliferation through generation of intracellular ROS and
lysates of heart muscle indicating that AZL plays protective activation of redox signaling pathways. Cell proliferation was
role in cardiac apoptosis. increased in a dose-dependent manner by high doses of INS
It has been previously observed that azelnidipine inhibits (0.110 M) but was inhibited by 0.1 M AZL. Namely, the
tumor necrosis factor-alpha-induced endothelial cell (EC) INS-increased phosphorylation of mitogen activated protein
oxidative stress through its AO properties (Nakamura et kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK
al. [220]). Azelnidipine, but not nitrendipine, completely 1/2) was inhibited by 0.1 M AZL. The same AZL concen-
inhibits the Ang II-induced ROS generation in ECs (Matsui tration blocked intracellular ROS production more effec-
et al. [221]). tively than 0.1 M nifedipine. The NADPH oxidase inhibitor,
Furthermore, azelnidipine, but not nitrendipine, was apocynin (0.010.1 M), prevented INS-induced mesangial
found to partially restore decreased pigment epithelium- cell proliferation. So, azelnidipine inhibits insulin-induced
derived factor (PEDF) mRNA levels in Ang II-exposed ECs. mesangial cell proliferation by inhibiting the production
This study suggests that AZL influence depends on its antiox- of ROS. Therefore azelnidipine may have the potential to
idative properties. Authors concluded that upregulation of protect against the onset of diabetic nephropathy and slow
PEDF by azelnidipine may become a therapeutic target its progression.
for the treatment of diabetic retinopathy associated with Azelnidipine inhibited H2 O2 -induced cell death in
hypertension. neonatal rat cardiomyocytes (Koyama et al. [227]). Azelnidip-
Antihypertensive agents with AO effects are potentially ine and nifedipine did not affect the H2 O2 -induced activation
useful for diabetic patients with hypertension. While DHP of extracellular signal-regulated protein kinases (ERK) and
type CA are among the most frequently used antihypertensive p38 MAPK (mitogen-activated protein kinase). In contrast,
drugs, azelnidipine has been reported to have a unique AO azelnidipine, but not nifedipine, inhibited H2 O2 -induced c-
effect in vitro and in vivo, in experimental animals (Ohmura Jun NH2 -terminal kinases (JNK) activation. Authors con-
et al. [222]). In hypertensive diabetic patients, azelnidipine cluded that azelnidipine has inhibited the H2 O2 -induced JNK
treatment for 12 weeks induced a more significant decrease activation and cardiac cell death. Therefore azelnidipine may
in erythrocyte LOOH level than amlodipine, although the have cardioprotective effects against OS.
values related to blood pressure during each treatment A specific atheroprotection activity of azelnidipine relates
remained comparable. These data confirm the usefulness of to inhibition of TNF--induced activator protein-1 activa-
LOOH level in erythrocyte membrane as a marker of OS in tion and interleukin-8 expression in human umbilical vein
vivo and indicate that azelnidipine has a unique antioxidative endothelial cells (HUVEC), through suppression of NADPH
property in humans. oxidase-mediated reactive oxygen species generation (Naka-
Daikuhara et al. [223] reported the results of the OLCA mura et al. [220]). TNF- could play a central role in patho-
study, based on combination of (1) olmesartan and a calcium genesis of insulin resistance and accelerated atherosclerosis in
channel blocker (azelnidipine) or (2) candesartan and a CCB the metabolic syndrome. The concentration of AZL found to
amlodipine in two groups of diabetic hypertensive patients. be effective in these in vitro experiments is within therapeutic
Patients treated with the first combination presented highly range. As EC do not possess voltage-operated L-type calcium
persistent early morning antihypertensive effect and stronger channels, it is suggested that the beneficial effects of azelni-
decrease in heart rate, fasting blood glucose and HbA1c levels, dipine are not likely due to CA property but to its unique AO
ability. Furthermore, it has been recently found that serum p22phox mRNA production, could play a role in its protective
levels of monocyte chemoattractant protein-1, a biomarker mechanism against OS.
for subclinical atherosclerosis, were significantly decreased by
the AZL treatment in patients with essential hypertension. In 3.5.9. Mebudipine. The protective effect of mebudipine (1,4-
this paper [220], authors hypothesize that, due to its unique dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicar-
TNF- signal modulatory activity and antioxidative property, boxylic acid 3-methyl-5-tert-butyl ester; BAY-n-6391) was
azelnidipine may be a promising DHP for targeting diabetes revealed on OS and LP (MDA decrease, SOD, GPX, and
and cardiovascular diseases in hypertensive patients with catalase increase) in myocardial ischemic-reperfusion injury
metabolic syndrome. in male rats (Ghyasi et al. [231]).
Shinomiya et al. [228] evaluated its AOA in cultured There are articles about other commercial and experi-
human arterial EC, under OS. Azelnidipine has shown a mental DHPs on OS, but we have reviewed only the most
potent antioxidative effect that could be of significant clinical commonly studied compounds. Effects of other commercial
benefit when combined with its long-lasting antihypertensive CA DHPs on OS are also mentioned in several parts of this
action and low incidence of tachycardia. review.
Azelnidipine inhibited TGF-1 and angiotensin II- (Ang
II-) activated 1(I) collagen mRNA expression in hepatic 3.6. 1,4-DHPs: Ca Agonists and Their AOA and ARA. For the
stellate cells (HSCs) (Ohyama et al. [229]). Furthermore, most popular calcium agonist DHP Bay K 8644 no reaction
TGF-1- and Ang II-induced OS and TGF-1-induced p38 with peroxyl radicals was registered (Toniolo et al. [114]).
and JNK phosphorylation were reduced in HSCs treated with However, interaction with other compounds possessing AOA
AZL. Azelnidipine significantly decreased inflammatory cell and ARA (quercetin) was found.
infiltration, profibrotic genes expression, HSC activation, LP, Opposite to that, AO N-acetylcysteine (NAC) diminished
oxidative DNA damage, and fibrosis in liver of CCl4 - or increase in Ca2+ transient amplitude and cell shortening
TAA-treated mice. Finally, AZL prevented decrease of the induced by ISO and forskolin, whereas NAC had no effect
expression of some AO enzymes and accelerated regression on the (S)-()-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-
of liver fibrosis in CCl4 -treated mice. Hence, the antifibrotic (2-trifluoromethylphenyl)pyridine-5-carboxylate()-Bay
mechanism of AZL against CCl4 -induced liver fibrosis in K 8644-induced increases (Andersson et al. [232]).
mice may have been due to an increased level of AO defense. Increased vasoconstriction responses to Bay K 8644 (3
As azelnidipine is widely used in clinical practice without 107 3 105 M) were significantly decreased by pyridoindole
serious adverse effects, it may provide an effective new antioxidant stobadine treatment in diabetes (Ceylan-Isik et
strategy for antifibrotic therapy. al. [233]).
The functional interaction between two L-type Ca2+
3.5.8. Manidipine. Manidipine, (2-[4-(diphenylmethyl)pip- channel activators, quercetin and Bay K 8644, has been
erazin-1-yl]ethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4- investigated in vascular smooth muscle cells. Biological ARA
dihydropyridine-3,5-dicarboxylate), is a DHP CCB with compound quercetin at nutritionally meaningful concen-
reported nephroprotective properties. Calo et al. [230] stud- trations limited the responsiveness of vascular L-type Ca2+
ied effect of manidipine on gene expression and protein channels to the pharmacological stimulation operated by Bay
level of OS related proteins: p22(phox) (human neutrophil K 8644. These data contribute to a better understanding of
cytochrome b light chain (CYBA)) and heme oxygenase-1, quercetin effects on experimental in vivo cardioprotection
HO-1. Relevance for antihypertensive effects was revealed. (Saponara et al. [234]). Thus, these findings indicated that
The study assessed the effect of manidipine on normal although Bay K 8644 does not exert potent and direct AOA,
subjects monocyte gene and protein expression of OS related yet acting as calcium agonist it may affect efficiency of AO
proteins such as p22phox, a NADPH oxidase system subunit, substances and vice versa.
critical in generating O2 , and HO-1, induced by and pro- Interaction of grapefruit juice (containing quercetin and
tective against OS. Manidipine was compared with the ACE its analogues) with DHPs CA diminished effectiveness of CA
inhibitor captopril and the CCB nifedipine, in the presence action of DHPs (Sica [235]).
and in the absence of sodium arsenite (NaAsO2 ) as an inducer
of OS. Monocyte p22phox (CYBA) mRNA production was 3.7. Interpretations of the Mechanism(s) of Radical
reduced by both manidipine and captopril, while no changes Scavenging and AOA by DHPs
were induced by nifedipine. Manidipine increased monocyte
HO-1 mRNA production, while nifedipine and captopril 3.7.1. Molecular Mechanism(s) of Radical Scavenging and
showed no effect. The effects of manidipine on p22phox AOA of DHPs in the Model Systems. 3,5-Dicarbonyl-1,4-di-
and HO-1 gene expression in the presence of OS were also hydropyridine derivatives possess powerful bis--carbony-
confirmed at the protein level. Thus, manidipine seems to lvinyl-amino conjugation and for that reason cannot be con-
suppress p22phox and to increase the HO-1 mRNA produc- sidered as ordinary amino antioxidants. The electron and/or
tion and protein level. The manidipine-induced increase of H donation from DHPs ensures their reductant role and
HO-1 gene and protein expression seems to be a peculiar results in AOA and ARA. Oxidation reaction from DHPs
effect of this drug since it is not observed with captopril results in production of corresponding heteroaromatic pyri-
and nifedipine. This effect, together with the reduction of dine derivatives.
Detailed studies were made about substituent in DHP dihydropyridine ring, that is, the presence of the hydrogen in
ring positions: 1,4-, namely, 4-unsubstituted-; 4-substituted: 1-position.
4-alkyl-; 4-aryl-; 4-alkylaryl- a.o.; 2,6-; 3,5- (diacetyl or Yanez et al. [197] have studied the reactivity of 11
dialkoxycarbonyl chain a.o.) electronic and steric effects on derivatives of 1,4-DHPs (including commercial CA) with
AOA and ARA of DHPs [44, 45, 51], see Sections 3.3.1 alkylperoxyl radicals and ABTS radical cation. The tested
and 3.5. The bell-shaped dependence of DHPs AOA on 1,4-DHPs were 8.3-fold more reactive towards alkylperoxyl
the 3,5-dialkoxycarbonyl- chain length was observed [44, radicals than to the ABTS cation radical. All commercial
45, 107, 109, 112], with the maximum activity at C2 H5 1,4-DHP type CCBs were weaker than Trolox-C. The par-
C4 H9 . Decrease of AOA and incorporation into liposomes for ticipation of the hydrogen atom in the 1-position appears
DHPs with alkyl chains longer than R > C4 H9 further were to be very relevant for exhibited reactivity. Hantzsch ester
clarified as probable tendency to self-aggregation of these (diludine) was again found to be the most active compound in
compounds ([51] and citation number 245 therein). Electron the reaction with alkylperoxyl radicals, 2.3-fold more active
acceptor/electron donor properties are relevant for expres- than Trolox. The photodegradation product of nifedipine
sion of AOA or ARA of 3,5-disubstituted DHPs. 3,5-Diacyl- (nitrosophenyl derivative of pyridine) also showed a high
substituted and 3,5-dicarbanilido- and 3,5-dipyridylamido- activity. Kinetic rate constants for the reaction between 1,4-
substituted DHPs are more active as AOs as their 3,5- DHP compounds and alkylperoxyl radicals exhibited a fairly
dicyano-substituted analogues, which have electron acceptor good linear correlation with the oxidation peak potential
properties [186]. of DHP derivatives. However, the activity of tested 1,4-
Dubur et al. [89] observed overwhelming steric influence DHPs towards ABTS radical cation showed an independence
of substituents in position 4 of the DHP ring. Gaviraghi between kinetic rate constants and oxidation peak potentials.
et al. [163, 164] proposed that AO activity of DHPs is Kirule et al. [195] and Tirzit et al. [196] studied mechanism
partly dependent on capacity of the 1,4-DHP ring to donate of AOA of 4-nitrophenyl-1,4-DHPs, nifedipine and its ana-
electrons to the propagating radical (ROO or RO ) and to logues, involving formation of 4-(2 -nitrosophenyl)-pyridine
reduce it to a less reactive form. The abstraction (donation) derivative (as active principle) as a result of intramolecular
of electron and/or H in the oxidation and LP reactions redox reaction, using chemical, electrochemical, and bio-
takes place from all 3,5-dicarbonyl-1,4-DHP systems and chemical approaches (see Sections 3.3.1 and 3.5.1).
results in the formation of corresponding pyridine derivatives Nunez-Vergara et al. [237] reported the electrochemical
(Augustyniak et al. [51]). The physicochemical mechanism of oxidation of C4-hydroxyphenyl-substituted 1,4-DHP deriva-
ARA and AOA of 1,4-DHP has been extensively studied and tives. The oxidation proceeds via formation of the unstable
discussed (Mulder et al. [236]), but precise mechanisms of dihydropyridyl radical, as confirmed by controlled-potential
their activity need further clarification. electrolysis (CPE) and ESR experiments. This type of 1,4-
The reactivity of C-4 substituted 1,4-DHPs possessing DHPs has significant activity towards the radicals even when
either secondary or tertiary nitrogen atom in the DHP ring compared with commercial 1,4-DHP drugs with well-known
toward alkyl, alkylperoxyl radicals, and ABTS radical cation antioxidant ability.
was determined in aqueous media at pH 7.4 [103]. These It was observed that nicardipine preferentially targets RO
compounds reacted faster toward alkylperoxyl radicals and radicals and is inactive against ROO . Lacidipine, on the
ABTS radical cation than alkyl ones. N-Ethyl-substituted other hand, is equally active towards both types of radicals
DHPs showed the lowest reactivity. (Gaviraghi et al. [164]). The cytoprotective effect against
The 4-methyl substituted DHP was the most reac- exposure to H2 O2 was more significant for lacidipine (ID50 =
tive compound in previously mentioned reactions (Lopez- 14 nM, its log = 5.4, membrane partition = 136000, assumes
Alarcon et al. [103]). However, it was less active (0.68 position located 7 A near to the membrane center; other
versus 1.0) than Trolox-C. DHPs having electron-donating less lipophilic DHPs located 1216 A far from the center)
substituents (4-Me-DHP and p-MeO-Phe-DHP) showed the as compared to amlodipine, nifedipine, and nicardipine, in
highest kinetic rate constants toward ABTS radical cation; p- smooth muscle cell culture (Gaviraghi et al. [164]). Oxidative
nitro-Phe-DHP, a compound with an electron-withdrawing effect of H2 O2 shifts the Ca channel toward an open state.
substituent, showed a lower kinetic rate constant; and N- Thus, the redox property of CCBs DHPs may augment their
alkyl-DHP compounds show kinetic rate constants lower CCB properties.
than the -NH-DHP. Oxidation of pharmacologically active Hantzsch 1,4-
Hydrogen at the 1-position of the DHP ring was revealed, dihydropyridines was found by electrogenerated superoxide,
according to the deuterium kinetic isotope effect studies, to be using a voltammetric approach in DMSO solutions (Ortiz et
involved in the proposed ARA mechanism. This fact is mostly al. [238] and Ortiz et al. [239]). Raghuvanshi and Singh [240]
noticeable in the case of alkyl radicals. N-Ethyl-substituted have also reported oxidative aromatization of these DHPs,
DHPs show the lowest reactivity when compared to Trolox induced by superoxide.
or nisoldipine. In all cases, the respective pyridine derivative Chemiluminescence (CL) was used in the studies ana-
was detected as the main product of the reaction (Lopez- lyzing the antioxidant activity of 12 various 4-flavonil-1,4-
Alarcon et al. [103]). Authors indicate that the kinetic rate dihydropyridine derivatives (Kruk et al. [241]) on a chemical
constants toward alkyl, alkylperoxyl, and ABTS radical cation system involving a superoxide radical anion. These deriva-
depend on the nature of the substituents in the C-4 position tives showed structural similarity to flavonoids, with respect
of DHP and the presence of the secondary amine group in the to the presence of rings A, B, and C. The results obtained in
this study indicate that the tested derivatives may catalyze for the same series of compounds. The trends in the peroxyl
conversion of superoxide radicals, through mimicking the and DPPH rate constants were found to be similar [236].
activity of superoxide dismutase by delivering H+ for reac- Tirzit et al. [242] have observed quenching of singlet
tion: oxygen by DHPs. This observation paved the ground for
further research related to reactions of DHPs with hydroxyl
O2 + O2 + 2H+ H2 O2 + 1 O2 (3) radicals (Tirzit et al. [243]), singlet oxygen (Kazush et
The enhanced emission of the light in the presence of al. [94]), and mechanisms of action. A series of 1,4-DHP
tested compounds was significant and related to stimulated derivatives in NAD-H-Cu2+ -H2 O2 system inhibited forming
of the hydroxyl radical (HO ), while 1,4-DHP derivatives
production of H2 O2 and 1 O2 from O2 . The latter species were
with electron donor substituents in the molecule were shown
removed from the reaction mixture by the following sequence
to be capable themselves of generating HO in the presence
of reactions:
of Cu2+ and H2 O2 . Rubene et al. [186] also described
H+ interaction of 1,4-DHP derivatives with Fentons reagent,
which produces hydroxyl radical (HO ). Rate constants of
O2 HO2 the DHPs reaction (1st order) with HO radical were high:
in the range 109 L mol sec1 , close to that of NADH,
cysteine and thiourea. 3,5-Diacetyl- derivatives reacted faster
HO2 + O2 HO2 + 1 O2 (4) compared to 3,5-dialkoxycarbonyl- ones. The reaction rate
decrease was observed in the case of substitution at position
2HO2 H2 O2 + 1 O2 4 as compared to 4-unsubstituted DHPs. Some DHPs having
electron donor -COO groups in the 3,5- or 2,6- positions
2 (1 O2 ) (O2 )2 + h] of DHP ring reacted immediately (having rate constants
higher as 109 L mol sec1 ). Rate constants with HO2
or to take part in spontaneous dismutation of H2 O2 : and O2 radicals were of lower degree. Thus DHPs acting
as oxygen radical scavengers could effectively inhibit ROS
2H2 O2 2H2 O + 1 O2 (5) related reactions of LP initiation stage.
The authors have offered an original concept of action for 4- Nifedipine and nitrendipine reactivity toward singlet
flavonil-1,4-dihydropyridine derivatives unrelated to that of oxygen was also studied [244]. Nifedipine was shown to
O2 radical-trapping, chain-breaking antioxidants. Instead, be a good scavenger of excited oxygen, mainly via physical
they showed that these compounds act similar to superoxide deactivation with values of the total rate constant ranging
dismutases, converting O2 to H2 O2 . Hydrogen peroxide from 20.8 105 M1 s1 (in dioxane) to 93.0 105 M1 s1
is less toxic for cells than O2 because it is predominantly (in propylene carbonate). The less reactive pathway generated
removed by peroxidases and catalases. Finally, AO effect of a photooxidation product. For that reason, a mechanism
these DHPs differed from those mediated by flavonoids with involving a perepoxide-like encounter complex in the first
a catechol structure of ring B, which are well-known 1 O2 step of the reaction path was proposed (see [244], Figures
quenchers. 8 and 9 therein). The dependence was observed on solvent
Mulder and collaborators came to similar conclusions, microscopic parameters of the total rate constant for the reac-
especially related to molecular mechanisms of antioxidative tion between singlet oxygen and 1,4-DHPs. These findings
activity of DHPs [236]. The AO properties of Hantzsch 1,4-di- show that nifedipine possesses stronger protective activity in
hydropyridine esters and two dibenzo-1,4-dihydropyridines, biological systems than nitrendipine.
9,10-dihydroacridine (DHAC) and N-methyl-9,10-dihydro- Density-functional-theory (DFT) calculations made by
acridine (N-Me-DHAC), have been explored by determin- Wang et al. [245] confirmed the former experimental obser-
ing the autoxidation of styrene or cumene at 30 C. These vations that Hantzsch ester, diludine, is potent antioxi-
experiments showed that Hantzsch esters are virtually inac- dant with high H atom-donating ability and relatively low
tive as chain-breaking antioxidants (CB-AOs), contrary to prooxidant activity. Possible reaction pathways for radicals
the findings observed by Lopez-Alarcon et al. [103] who derived from 1,4-dihydropyridine and the resonance modes
used CB-AOA in aqueous media at pH 7.4. Their reactivity for radical species were given [245].
toward peroxyl radicals was shown to be some 5 orders of Moreover, two ethoxycarbonyl (EtOCO) substituents
magnitude lower than that of the excellent CB-AO, 2,2,5,7,8- at C(2) and C(6) should further enhance Hantzsch ester,
pentamethyl-6-hydroxy-chroman (PMHC). diludine H-atom-donating ability due to resonance effects.
DHAC was found to be 10 times less reactive than However, DHPs should be used in nonpolar rather than in
PMHC kinetic measurements using DHAC, N-deuterio- polar solvents since, in the latter, not H-atom but electron
DHAC, and N-Me-DHAC, pointing out the abstraction of transfer is preferred in the radical scavenging process [245].
N-H hydrogen in DHAC by peroxyl radicals, despite the fact Mulder et al. [246] proposed that quantum-thermochem-
that the calculated C-H bond dissociation enthalpy (BDE) in ical calculations must be used with caution to indicate a
DHAC is about 11 kcal/mol lower than the N-H BDE. The promising lead antioxidant, as they criticized the density-
rates of hydrogen atom abstraction by the 2,2-diphenyl-1- functional-theory (DFT) calculations made by Wang et al.
picrylhydrazyl radical (DPPH ) have also been determined [245].
3.7.2. Possible Mechanisms of DHPs ARA and AOA in the Bio- 3.8. DHPs: Anti- or Prooxidants? Several substances (ascorbic
logical Systems: Interaction with Other OS Modifiers. Some of acid being the most commonly studied) can serve either as
these mechanisms were already described (Sections 3.3.1 (2)- antioxidants or as prooxidants, depending on given condi-
(b); 3.3.1 (2)-(c); 3.3.1 (3)-(b); 3.5). tions (Herbert [250]). Therefore, Halliwell [251] has reported
Enzymatic sources of ROS with confirmed functional role dilemma related to polyphenols as possible antioxidants
in hypertension are NADPH oxidase, NO synthase (NOS), and prooxidants, causing experimental artifacts (about 25)
xanthine oxidase, and cyclooxygenase. Godfraind [3] has by oxidation of antioxidant compounds in the cell culture
reviewed AO effects and protective action of calcium channel media. Nevertheless, it is generally accepted opinion that
blockers (CCBs). Yao et al. [247] observed antioxidant effects polyphenols act as antioxidants in vivo. Studies on DHPs also
(as inhibition of LP) for cardio- and neuroprotective CCBs face such a dilemma. The exact roles (anti- or prooxidative) of
(3300 mol/L), including 7 DHPs, in homogenates of rat any specific DHP depend on its structure, applied/achieved
brain. IC50 values (M) were as follows: nifedipine (51.5) > concentration, and specificity of the target/experimental
barnidipine (58.6) > benidipine (71.2) > nicardipine (129.3) testing system.
> amlodipine (135.5) > nilvadipine (167.3) > nitrendipine This situation resembles the case of antioxidative effects
(252.1) > diltiazem (>300) = verapamil (>300). There are of tocopherol, which depends on the fate of the secondary
also research articles describing the AO properties of CCBs radical as proposed by Winterbourn [252]. The question was
through direct scavenging effect or through preservation Vitamin E - Pro- or Antioxidant?:
of the endogenous SOD activity. These findings indicate Antioxidant:
that CCBs may also act by reducing the production of
vasoconstrictors, angiotensin, and endothelin. LOO + Toc
When present in concentrations that can be achieved
in plasma, CCBs may inhibit LP formation [3]. This AO LOOH + Toc (Toc = -tocopheryl radical)
(6)
activity seems to be typical for high lipophilic CCBs because Toc + LOO
their chemical structure facilitates proton-donating and
resonance-stabilization mechanisms that quench the free chain termination of lipid peroxidation
radical reaction. Their insertion in the membrane, near
polyunsaturated fatty acids at relatively high concentrations, Prooxidant:
potentiates proton donation (or atomary H) to lipid peroxide
molecules, thereby blocking the peroxidation process. The Toc + Lipid-H Lipid (in LDL particles) (7)
remaining unpaired free electron associated with the CCB
molecule can be stabilized in well-defined resonance struc- This example shows that, generally speaking, any AO must
tures associated with the DHP ring (Mason et al. [48]). fulfil several criteria to be considered as an effective com-
The radical reaction (according to Godfraind [3]) that pound physiologically:
describes the AO effects of a DHP CCBs is LOO + DHP
(i) It must be able to functionally interact with endoge-
LOOH + DHP (where LOO is lipid peroxide radical),
nous scavengers, even at low concentrations.
which in general is reaction (7) of the LP reaction cascade
consisting of 10 reactions (Scheme 2) [89]. (ii) It must affect endogenous pathways of OS.
As the rate constants of in vitro interaction of 4- (iii) It should not have undesirable adverse effect.
substituted DHPs with peroxyl radicals are three orders
of magnitude lower than that of the vitamin E derivative, (iv) It should manifest the antioxidant efficacy dependent
these DHPs must be considered as weak AO (Ursini [204]). on the oxidant.
However, due to partition coefficient of DHPs in membranes (v) It must discriminate among different strategies
and in case of specific binding, high local concentration of needed for 1-electron and 2-electron processes.
DHPs may be obtained. (vi) Radical scavengers can be prooxidant unless linked to
DHPs without CCB properties, for instance Bay w 9798, a radical sink (Winterbourn [252]).
although structurally related to nifedipine, inhibit TNF-
-induced vascular cell adhesion molecule-1 expression in According to these statements, DHPs could be effective as
endothelial cells by suppressing reactive oxygen species AO under physiological conditions in vivo (Godfraind [3]
generation [248]. and others [30, 31, 38]) and in vitro in various experimental
Mitrega et al. [249] have discovered that antiarrhythmic systems (cell and tissue) (see Sections 3.3; 3.4; 3.5).
and hemodynamic effects of oxidized heteroaromatic DHPs, Hence, calcium antagonists appeared to disrupt the
oxy nifedipine, oxy nimodipine, oxy nitrendipine, and oxy fine balance between the production and scavenging of
nisoldipine, suggest that CCB DHPs and their metabolites ROS. Nifedipine, verapamil, and diltiazem were shown to
could act at least in two ways: targeting OS related events induce significant oxidative stress in the epididymal sperm
as reductants (see Section 3.5.1 (1)) and/or bypassing OS (increased MDA and decreased catalase and superoxide
related metabolic routes. Authors postulated that, contrary to dismutase activity). This may be the reason for the induction
current belief, NIF metabolites are pharmacologically active. of male infertility [253].
ATP sensitive potassium channels were mentioned as a tar- The dualism of 1,4-DHP effects has been recorded as
get. inhibition or promotion of LP, as quenching or initiating
oxygen and nitrogen free radical chain reactions, radiopro- cycles of redox and cell functions (the electroplasmic cycle)
tecting or radiosensitizing, antagonizing or synergizing Ca2+ (Wagner et al. [258]).
in electromechanical coupling, as well as in the membrane
stabilization or labilization.
4. Conclusions
3.9. Could DHPs Be Involved in Antioxidative Stress? Before 1,4-Dihydropyridines (1,4-DHPs) have broad spectrum of
being applied in vivo, the optimal dose and optimal time OS modulating activities. DHPs have reducing and lipid
intervals for DHPs application must be known. Namely, while peroxidation inhibitor properties, act as reductants in simple
ROS have been traditionally considered as toxic byproducts chemical systems, and stabilize various biological systems
of aerobic metabolism, we know nowadays that ROS may act (LDL, mitochondria, microsomes, cells, and tissues) against
as essential signaling molecules, needed for the control of OS. Examples and peculiarities and mechanisms of antiox-
many different physiological processes. Whether the role of idant activity (AOA) and antiradical activity (ARA) as well
ROS will be damaging, protective, or signaling depends on as stress-protective effect of DHPs including commercial
the delicate equilibrium between time- and location-specific calcium antagonists (CA) were highlighted. These activities
ROS production and scavenging. Accordingly, the imbalance depend on various structural parameters related to DHPs
of the increased AO potential, so-called antioxidative stress, (presence and character of substituents), lipophilicity, and
could be dangerous similar to chronic OS, in particular in depth of the incorporation in the biological membranes. They
case of extended exposure. Inappropriate interventions in also depend on the experimental model system for exploring
the oxidative homeostasis by excessive antioxidants especially the lipid peroxidation or oxidative stress. Stress-protective
in case of chronic exposure to antioxidants might have effect of some metabolites of CA (nifedipine) is reviewed.
very negative effects as was published in the ATBC study, Although some DHPs, including CA, have prooxidant prop-
erties (on epididymal sperm cells), they can generally be
showing an increased cancer incidence in smokers treated
considered as potent antioxidants. Therefore, comparison
by excessive beta-carotene [254]. Therefore, overconsump-
of the AOA and ARA of different DHPs (mutually and
tion of any natural or synthetic AO, including DHPs, as
with other AOs) was described in detail. According to the
dietary supplements or drugs, must be avoided in order to data presented, the DHPs might be considered as bellwether
suppress oxidative damage and must not disrupt the well- among synthetic compounds targeting OS and as a kind of
integrated antioxidant defense network (Poljsak [255] and pharmacological measure for respective field of organic and
Poljsak and Milisav [256]). This is especially important when medicinal chemistry.
administrating lipid-soluble antioxidants that can be stored
in biomembranes, thus not only attenuating or preventing LP
but also affecting physiological effects of the natural antiox-
Abbreviations
idants, in particular tocopherol. The interrelationship with AD: Alzheimer disease
the status of endogenous antioxidants/prooxidants should be AO(s): Antioxidant(s)
followed. AOA: Antioxidant activity
DHPs primarily suppress the initiation stages of LP ARA: Antiradical activity
process. They do not entirely stop the LP propagation. Acting CA: Calcium antagonist(s)
synergistically with tocopherol, diludine may prevent patho- CCB: Calcium channel blocker
logical excess of ROS production within the lipid moiety of DHP(s):Dihydropyridine(s)
the cellular membranes and LDL. However, due to its low DNA: Deoxyribonucleic acid
solubility and fast metabolism, its concentration in the cells HEH: Hantzsch ester
is low. For that reason, it cannot cause antioxidative stress, 4-HNE: 4-Hydroxy-2-nonenal
even if used for an extended period of time. Thus diludine IMAC: Inner membrane anion channel
(diethone) could be only a mild antioxidant; it has potential Lox: Lipoxygenase
for restoring the pool of natural antioxidants (as synergist of LP: Lipid peroxidation
-tocopherol and polyphenols) in the cells. MDA: Malonyl dialdehyde
Moreover, DHPs CA used for cardioprotection and Mit: Mitochondria
vasodilatation as commercial drugs in low concentrations NADH: Reduced nicotinamide adenine
are fast metabolized via CYP3A4, and for that reason their dinucleotide
application does not induce cellular AO stress [53, 160]. NADPH: Reduced nicotinamide adenine
However Godfraind and Salomone [257] have postulated no dinucleotide phosphate
evidence that allows recommending dietary supplementation NO: Nitrogen oxide
with antioxidants for the primary or secondary prevention of OS: Oxidative stress
cardiovascular disease. PD: Parkinsons disease
So far, there are no reports on antioxidative stress caused RNS: Reactive nitrogen species
by some DHPs, diludine and its analogues. Diludine and ROS: Reactive oxygen species
its analogues therefore could act as adaptogens supporting SOD: Superoxide dismutase
hormetic effects of mild oxidative stress. These compounds TBARS: Thiobarbituric acid reactive substances
may act as potential multisided modulators of Yin-Yang TG: Triglycerides.
Conflict of Interests on human health, Current Medicinal Chemistry, vol. 20, no. 37,
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http://dx.doi.org/10.1155/2016/4251912
Review Article
Redox Control of Multidrug Resistance and
Its Possible Modulation by Antioxidants
Aysegul Cort,1 Tomris Ozben,2 Luciano Saso,3 Chiara De Luca,4 and Liudmila Korkina5
1
Department of Nutrition and Dietetics, Faculty of Health Sciences, Sanko University, Incili Pnar,
Gazi Muhtar Pasa Bulvar, Sehitkamil, 27090 Gaziantep, Turkey
2
Department of Biochemistry, Akdeniz University Medical Faculty, Campus, Dumlupnar Street, 07070 Antalya, Turkey
3
Department of Physiology and Pharmacology Vittorio Erspamer, La Sapienza University of Rome,
Piazzale Aldo Moro 5, 00185 Rome, Italy
4
Evidence-Based Well-Being (EB-WB) Ltd., 31 Alt-Stralau, 10245 Berlin, Germany
5
Centre of Innovative Biotechnological Investigations Nanolab, 197 Vernadskogo Prospekt, Moscow 119571, Russia
Correspondence should be addressed to Liudmila Korkina; korkina@cibi-nanolab.com
Received 12 August 2015; Revised 14 November 2015; Accepted 18 November 2015
Academic Editor: Sidhartha D. Ray
Copyright 2016 Aysegul Cort et al. This is an open access article distributed under the Creative Commons Attribution License,
Clinical efficacy of anticancer chemotherapies is dramatically hampered by multidrug resistance (MDR) dependent on inherited
traits, acquired defence against toxins, and adaptive mechanisms mounting in tumours. There is overwhelming evidence that
molecular events leading to MDR are regulated by redox mechanisms. For example, chemotherapeutics which overrun the first
obstacle of redox-regulated cellular uptake channels (MDR1, MDR2, and MDR3) induce a concerted action of phase I/II metabolic
enzymes with a temporal redox-regulated axis. This results in rapid metabolic transformation and elimination of a toxin. This
metabolic axis is tightly interconnected with the inducible Nrf2-linked pathway, a key switch-on mechanism for upregulation
of endogenous antioxidant enzymes and detoxifying systems. As a result, chemotherapeutics and cytotoxic by-products of their
metabolism (ROS, hydroperoxides, and aldehydes) are inactivated and MDR occurs. On the other hand, tumour cells are capable
of mounting an adaptive antioxidant response against ROS produced by chemotherapeutics and host immune cells. The multiple
redox-dependent mechanisms involved in MDR prompted suggesting redox-active drugs (antioxidants and prooxidants) or
inhibitors of inducible antioxidant defence as a novel approach to diminish MDR. Pitfalls and progress in this direction are
discussed.
1. Introduction cells [2]. Frustrating the great expectations raised, ABC

transporter/modulators/reversals proved to have insufficient
It is common knowledge that multiple drug resistance (MDR) clinical efficacy and very high toxicity. Novel biological
has crucial negative impact on the clinical outcomes of con- approaches have been recently developed in laboratory
ventional cytotoxic anticancer therapies and of those based to modulate ABC transporter-mediated MDR, including a
on specific drugs targeting molecular pathways implicated monoclonal antibody that binds specifically to P-gp, thus sup-
in cancer cell functions and survival strategies. Since the pressing drug transport, small interfering RNA technology
discovery of the first ATP-binding cassette (ABC) trans- to decrease the expression of ABCB1, antisense oligonucleot-
porter P-glycoprotein (P-gp), ABC drug transporters have ides, and agents attenuating P-gp transcription [3]. Though
become targets for improving anticancer chemotherapy. Up very promising, these biologicals are still lacking clinical
to now, more than 49 different ABC transporters have been proof-of-concept data.
found and cloned [1]. A majority of MDR modulators or In any case, the evident and numerous adverse effects of
reversals are themselves substrates of the transporters that MDR modulators stimulated additional studies on physio-
compete with anticancer agents for the efflux from tumour logical role(s) of MDR in the human organism. It has been
reported that MDR relies not exclusively on transporting activated by planar aromatic hydrocarbons of natural or
systems for drug uptake and efflux, but also on intracellular synthetic origin [1012]. A second group of chemical sensors
drug metabolism and DNA damage [4]. Transporting and comprises nuclear receptors, such as pregnane X, constitutive
metabolic systems defining MDR are expressed in the major- androstane, peroxisome proliferators-activated, liver-X, and
ity of normal cells, are essential for nutrients uptake and farnesoid-X receptors, recognising a wide variety of xeno-
metabolites efflux, and play a vital role in protecting cells and endobiotics [1214]. Nuclear factor erythroid-derived
against xenobiotics. Hence, harsh inhibition of a functionally 2-related factors (Nrf1 and Nrf2) and related capncollar-
essential mechanism results in general intoxication. (CNC-) basic leucine zipper proteins belong to another
To gain protection against foreign invasions and maintain family of sensors activated by oxidants and electrophiles
homeostasis, the human organism employs several types of [15, 16]. Activation of such recognition elements after ligand
physical, chemical, and biological defence systems. For exam- binding may result in alterations of ion channel conductivity,
ple, skin and other lining epithelia mechanically prevent inva- kinase machinery, and cytoplasmic and nuclear transcription
sion of relatively large organic and inorganic particles. The factors, inducing cell response (signal transduction).
immune system has been evolved to fight cellular invaders Signal transduction is often mediated by redox substances
and high-molecular-weight compounds of biological origin. (superoxide anion radical, hydrogen peroxide, lipid perox-
The chemical defence system, consisting of biosensoring, ides, aldehydes, and others) [1721]. At moderate concentra-
transmitting, and responsive elements, has been evolved, tions, they are signals to start gene transcription via activation
starting from primitive eukaryotes and lower plants [5], to of transcription factors (nuclear factor B (NF-B), activator
protect multicellular organisms against environmental chem- protein 1 (AP-1), and antioxidant response element- (ARE-)
ical insults (xenobiotics) and to maintain homeostasis of binding proteins) or initiating the protein kinase cascade [5,
endogenous low-molecular-weight metabolites (endobiotics) 16, 19]. The latter pathway leads to the interaction with specific
[6]. Being exposed to xenobiotic (drug) stress, an organism is ARE of DNA motifs on promoters of antioxidant defence
challenged to rapid and appropriate adaptation by activating enzymes such as GST, Mn-superoxide dismutase (MnSOD),
constitutive and expressing inducible systems, thus attenuat- and glutamyl-cysteine ligase, among others [15].
ing negative biological consequences. For this purpose, an Inherited or acquired alterations at any key point of the
array of gene families and molecular pathways have been chemical defence system might lead to chronic intoxication
developed during evolution to prevent cellular access, to and numerous human pathologies (chronic inflammation,
detoxify and eliminate toxins, and to repair chemical damage. degeneration, carcinogenesis, multiple chemical sensitivity
The active efflux proteins, for example, P-glycoproteins (P- syndrome, etc.) in the inherited or acquired MDR, respec-
gp) [7], multidrug resistance (MDR) proteins [8], and multi- tively (Figure 1). Usually, the course of anticancer chemo-
xenobiotic resistance (MXR) proteins [9], directly eliminate therapy induces the overexpression of drug transporters
slightly lipophilic organic xenobiotics from cells serving as MDR/MXR/P-gp [3, 4], activation of sensoring receptors,
the first line of chemical defence. Escaping the first-line electrophile/oxidant sensors, transcription factors, and over-
guardians, once in the cytoplasm, toxic nucleophilic com- expression/activation of detoxifying/antioxidant enzymes [1].
pounds undergo biotransformation by the oxidative phase Collectively, it causes rapid metabolism and elimination
I enzymes (cytochrome P450 (CYP), flavoprotein monooxy- of both the anticancer drugs and cytotoxic by-products
genase, hemeoxygenase, amine oxidases, xanthine oxi- targeting tumour cells. Since the chemical defence system is
dase, and others) to become electrophilic. The electrophile ubiquitous for all human organs and tissues and central to
is subjected to reductive or conjugative modification by organism functions, the attempts for its pharmacological sup-
phase II enzymes (glutathione-S-transferases (GSTs), UDP- pression in order to diminish MDR potentially bear the risk
glucuronosyltransferases (UGTs), catechol-O-methyl trans- of a multitude of undesired side effects. Upon the pharma-
ferases (COMT), N-acetyl transferases (NATs), and many cological interaction with components of universal chemical
others). defence system, the good guy evolved on purpose to protect
Reactive oxygen species (ROS) generated as by-products multicellular organisms from low-molecular-weight chemi-
of phase I reactions are rapidly reduced to nontoxic physi- cals could become a bad guy blocking desired therapeutical
ological levels by antioxidant enzymes (superoxide dismu- effects of anticancer drugs (MDR).
tases (SODs), catalase (CAT), glutathione peroxidases (GPx), On the grounds of our current knowledge, redox regu-
peroxiredoxins (PRx), and nonenzymatic antioxidants, such lation of multiple molecular pathways essential for human
as reduced glutathione (GSH), uric acid, ascorbic acid, and chemical defence system can be implicated differentially in
ceruloplasmin, among others). All these constitutive protec- normal host and in tumour cells. Owing to the fact that redox
tive systems are sufficient to cope with low levels of xenobi- balance in tumour cells is greatly altered as compared to
otics or endobiotics. The inducible chemical defence relies on that of normal host cells [22, 23], selective redox inhibitors
the array of stress responsive genes. In this case, chemical targeting tumour-associated chemical defence as a cause of
stressors like anticancer chemotherapeutics should first be MDR should be developed. Regarding potential health effects
recognised by specific sensors which, in turn, transmit alarm of redox modulators on tumours, they are mainly attributed
signals to activate or express de novo transporting, biotrans- to cancer chemoprevention, direct anticancer action (for
forming, and detoxifying enzymes. comprehensive review, see [23]), cancer sensitisation to con-
The primary member of mammalian proteins-sensors of ventional chemotherapeutics, preferentially through MDR
organic chemicals is the aryl hydrocarbon receptor (AhR), suppression/reversal, cancer sensitisation to radio- and
XB
P-gp MDR MXR
Metabolite Phase I EB
excretion (CYPs, HO1, and
Prx)
Phase II ROS
(GSTs, UGTs, COMT, NAT, etc.)
Antioxidant systems
ROS (SOD, CAT, GPx, and
Prx)
(a)
MDR
P-gp MXR
Anticancer drug
PI3-K
ROS AhR
Transcription
factors
NR
(NF-B, AP-1)
=
Nr Antioxidant enzyme
f2 induction
AR
E
Phase II enzyme
induction
Inflammatory
response
Phase I enzyme
induction
(b)
Figure 1: Inherited and acquired multiple drug resistance. (a) In the inherited multiple drug resistance (MDR), chronic exposure of
normal cells to low levels of unknown xenobiotics (XB) or/and endobiotics (EB) takes place. It causes upregulation of ATP-binding
cassette transporters such as P-glycoprotein (P-gp), MDR proteins (MDRs), and multiple xenobiotic resistance (MXR) without induction by
anticancer drugs. Single nucleotide polymorphisms of phase I and II metabolic enzymes and efflux transporters often accompany inherited
MDR and they could also be a causative reason for the resistance. Reactive oxygen species-mediated modulation of xenobiotics/drug
metabolism is similar to that in the acquired drug resistance. This cellular pattern seems to be associated with high risk of tumour
transformation. ROS: reactive oxygen species; MDR: multiple drug resistance transporters; MXR: multiple xenobiotic resistance transporters;
P-gp: P-glycoprotein; CYP: cytochrome P450; HO1: hemeoxygenase-1; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione
peroxidase; PI3K: phosphatidylinositol-3 kinase; AhR: aromatic hydrocarbon receptor; NF-B: nuclear factor kappa B; AP-1: activator
protein 1; NR: nuclear receptor; Nrf2: nuclear factor erythroid-derived 2-related factor 2; ARE: antioxidant responsive elements. (b) In the
acquired MDR, chemotherapeutics induce redox-dependent MDR expression and activity in tumour cells. Chemotherapeutics activate also
aromatic hydrocarbon receptor- (AhR-) driven and ROS-regulated expression of transcriptional factors (nuclear factor kappa B (NF-B) and
activator protein 1 (AP-1)) which initiate inflammatory response. Reactive oxygen species (ROS) mediate activation of phosphoinositol-3
kinase upstream of inflammatory cytokine transcription and synthesis. ROS and AhR-associated stimulation of Nrf2 followed by antioxidant
responsive element of DNA motif causes upregulation of protective, antioxidant, and detoxifying systems, such as antioxidant phase I and II
enzymes.
Cancer therapies
Cancer
Redox adjuvants
chemoprevention
Photodynamic
Chemotherapy therapy
Radiotherapy
Redox Redox
Sensitisation Redox
(synergy with
Direct a drug)
antitumour Sensitisation
action to Direct
radiotherapy photochemical
toxicity
MDR
suppression
Host tissues
protection
Figure 2: Redox-active substances and cancer. A variety of redox-active substances (direct or indirect antioxidants) are known to exhibit
cancer chemopreventive properties. In the pharmacological anticancer protocols, redox-active agents could be used as direct anticancer
chemotherapeutics or synergies with cytotoxic effects of conventional anticancer drugs. Here, we discuss the feasibility of such substances in
suppression/reversal of acquired MDR. The redox agents are often used for the protection of normal tissues/organs against toxic effects of
chemotherapy and radiotherapy.
photodynamic therapies, and protection of normal host regulations are similar to those characteristic for acquired/
organs/tissues against damage by chemo- and radiotherapy chemotherapy-induced MDR. In this line, the development
(Figure 2). of intrinsic MDR correlates with increased risk of carcino-
This review will discuss existing and perspective possibil- genesis, and the process is under network-like redox control
ities of differential targeted modulation of redox-dependent (for comprehensive review, see [1]). It appears that the
components/pathways of intrinsic and induced chemical classical paradigm of cancer chemoprevention with redox-
defence as an emerging strategy for combinatory anticancer active nontoxic substances could be interpreted in terms of
therapies to overcome MDR. Molecular pathways-targets for intrinsic MDR prevention. Furthermore, intrinsic MDR is
MDR attenuation or even reversal by redox-active substances a hallmark of stem cells, both normal and tumour, because
will be described in detail. high resistance to any toxin would guarantee survival and
maintenance of stem cell populations.
2. Intrinsic Multidrug Resistance
(MDR): Is It Possible to Overcome It by 2.2. Gene Polymorphisms Influencing Drug Metabolising
Redox Modulation? Enzymes May Result in Ultrafast Drug Elimination or
Extremely Slow Formation of Cytotoxic Redox By-Products.
2.1. Inherited Overexpression of Drug Transporters Accelerates The cytochrome P450 (CYP) system is a superfamily of
Drug Efflux from Target Cells. Some individuals possess isozymes, located in the smooth endoplasmic reticulum,
the so-called intrinsic MDR having never been exposed to mainly in the liver, but also in extrahepatic tissues (e.g.,
chemotherapy. Genetical predisposition to resist xenobiotic intestinal mucosa, lung, kidney, brain, lymphocytes, placenta,
stress could, in principle, be explained in terms of single and skin), involved in the biotransformation of numerous
nucleotide polymorphisms (SNPs) of complex MDR sys- lipophilic xenobiotics into more hydrophilic, less toxic, and
tem components, starting from drug transporters, censor- more easily excreted metabolites [11, 24, 25]. The major CYP
ing receptors, and xenobiotic/drug metabolising enzymes enzymes involved in human drug metabolism belong to fam-
(see several examples below). On the other hand, a lead- ilies 1, 2, and 3, the specific drug metabolising isoforms being
ing hypothesis indicates intrinsic MDR as a result of Cyp1A2, Cyp2C9, Cyp2C19, Cyp2D6, and Cyp3A4/3A5.
chronic (silent) exposure to low-level xenobiotic stressors Each CYP isoform is a product of specific gene. For some
or endogenous disturbances of lipid, glucose, and/or hor- isoforms, the existence of genetic polymorphisms has been
mone metabolism. Therefore, the molecular pathways of its demonstrated. The allelic variants may be due to the deletion
of the entire gene, SNPs, deletion or insertion of fragments of becomes a probable candidate to maintain cell stemness
DNA within the gene, or multiplication of gene copies, lead- [1]. Therefore, the development of clinically efficient redox
ing to absent, deficient, or enhanced enzyme activity. Thus, modulators of MDR should selectively target cancer stem
the population can be classified into Extensive Metabolisers cells, while leaving normal stem cells intact.
(EM, individuals with normal capacity), Poor Metabolisers
(PM, individuals with reduced/null metabolic activity), and
Ultrarapid Metabolisers (UM, individuals with a higher- 3. Redox Dependence of Acquired
than-normal metabolic activity). It seems that opposite pop- Multidrug Resistance: Modulation by
ulations of PM and UM could be at risk of constitutive Direct and Indirect Antioxidants
MDR, because UM would rapidly metabolise/excrete parent
molecules of anticancer drugs, while PM would not produce Most chemotherapeutic agents generate ROS, which bind to
ROS as by-products of anticancer drug metabolism. These specific structures within the cancer cells and promote cell
by-products possess strong cytotoxicity against cancer cells. death. Chemotherapeutic agents disturb the redox homeosta-
Hence, the routine clinical diagnostics based on determina- sis in cells and change their ability to cope with excessive ROS
tion of CYP SNPs produce a reliable prediction of individual levels through the production of protective direct antioxi-
chemosensitivity/chemoresistance/MDR to anticancer ther- dants [35]. Direct antioxidants are modified in this process
apies. Several polymorphisms have been connected with the and need to be resynthesised [36]. Glutathione (GSH) is con-
inducibility or enzymatic activity of the abovementioned sidered as the main redox buffer in a cell because it supplies
drug metabolising CYP isoforms [6]. large amounts (millimolar concentrations) of reducing equiv-
alents [37]. The intracellular thiol redox status is described as
2.3. Redox-Active Inhibitors of Drug Transporters and Recep- the ratio of reduced to oxidised forms of thiols (GSH/GSSG),
tors Associated with Drug Detoxifying Enzymes: Hopes and which decreases under oxidative stress conditions, and GSH
Reality. Notwithstanding the growing interest and great reversibly forms mixed disulfide bonds between protein
hopes for natural nontoxic redox agents to prevent/inhibit/ thiols (S-glutathionylation) to prevent protein oxidation [38].
reverse MDR ([26]; in this review), drug development Besides GSH, thioredoxin (Trx), another important endoge-
remains rather complicated due to low bioavailability, defined nous antioxidant, provides protection against oxidative stress
by restricted absorption through intestine, lining epithelia, [39, 40]. Nrf2 regulates Trx and sulfiredoxin enzymes, which
and skin [24, 25], rapid metabolism, and excretion. Absorbed are involved in the regeneration of the reduced form of
MDR inhibitors become themselves targets for the classical nicotinamide adenine dinucleotide phosphate (NADPH) and
pathways of xenobiotic detoxification/drug metabolism [27 synthesis of GSH [41]. Among the potential mediators of
29]. In phase I, they are predominantly metabolised by micro- chemoresistance, Trx plays critical roles in the regulation of
somal CYPs. Then, phase II glucuronidation by UGT [30], cellular redox homeostasis and redox-regulated chemoresis-
sulfation by phenol and catecholamine specific sulfotrans- tance [4244].
ferases (SULT1A1 and SULT1A3) [11], methylation by COMT In a recent study, Zhang et al. have shown that inhibiting
[31], and binding with glutathione through GST occur [12, Nrf2 expression through the transfection of shRNA plasmids
28]. in non-small-cell lung cancer cells significantly inhibited the
To improve candidate MDR inhibitor bioavailability and expressions of glutathione pathway genes, antioxidants, and
attenuate its metabolic disruption, several approaches have multidrug resistance proteins and induced the generation of
been implied such as injectable forms, other sophisticated ROS, decreased the level of GSH, and inhibited cell prolifera-
drug delivery systems, combination with adjuvants like piper- tion [45]. It has been reported that diffuse large B lymphoma
ine and caffeine to diminish glucuronidation, and chem- cells expressed higher-than-normal basal levels of Trx, which
ical modification of parent molecules to bypass efficient was associated with decreased survival. Suppressed Trx
metabolic guardians [6, 24, 27]. inhibited cell growth and clonogenicity and sensitised the
A very high probability of drug-drug interactions lymphoma cells to doxorubicin [44]. In a recent study,
between the adjuvant therapeutics for MDR inhibition and Raninga et al. [46] have reported the cytoprotective role of
anticancer therapies themselves, as inducers of MDR, should tTrx1 and thioredoxin reductase 1 (TrxR1) enzyme in multiple
also be taken into consideration [32]. It has been reported myeloma. Trx inhibitors were utilised in a variety of human
that potential MDR suppressors of herbal origin may easily cancers including acute myeloid leukemia [47], colorectal
interact with the same efflux (P-gp) and metabolic (Cyp3A4) cancer [48], and lung cancer [49] to inhibit tumour growth
pathways as anticancer agents do, resulting in opposite and to stimulate ROS-induced apoptosis. Auranofin, a TrxR1
outcomes: inhibition or expression of MDR components, inhibitor, caused oxidative stress-induced cytotoxicity and
depending on timing, dosages, posology, and route of drug apoptosis in cancers including chronic myeloid leukemia
administration [33]. Recent findings have shown that this [50], chronic lymphocytic leukemia [51], prostate cancer [52],
kind of drug-drug interaction is highly influenced by genetic and breast cancer [53]. Signal transducer and activator of
polymorphisms of efflux proteins (MDR1) and metabolic transcription 3 (STAT3) activation is commonly observed
enzymes (Cyp3A5) [34]. in multiple myeloma, chronic lymphocytic leukemia, gas-
Emerging evidence shows that MDR could be an evolu- tric cancer, lung cancer, and laryngeal carcinoma. Dietary
tionary defined mechanism to preserve normal and cancer gamma-tocotrienol inhibited both induced and constitutive
stem cell populations. In this direction, redox signalling activation of STAT3 in multiple myeloma and prostate cancer
cell lines [54]. High-dose intravenous ascorbate inhibited induced ROS production in human choriocarcinoma cells
NADPH-oxidase and was selectively toxic to tumours with by reducing BCRP and GSH content and activating Nrf2-
low CAT activity [55]. Recent study has reported that a naph- targeted genes: NAD(P)H dehydrogenase, quinone 1 (NQO1),
thoquinone derivative induced cell death depending on Bax and hemeoxygenase-1. On the other hand, genetic knockout
deficiency. In conclusion, it has been suggested that naphtho- of TrxR1 gene resulted in liver insensitivity to acetaminophen
quinone might be clinically feasible to overcome chemoresis- due to drastic disruption of the link between redox home-
tance [56]. ostasis and drug metabolism in the liver [72]. Glyoxalase 1,
Plant-origin polyphenols or their synthetic derivatives a key enzyme converting -oxoaldehydes into correspond-
have been recognised as redox-active molecules with rel- ing -hydroxy acids, has been found to be amplified in
atively low toxicity. Some of them, for example, luteolin, many primary tumours and cancer cell lines [73]. In this
apigenin, and chrysin, exert both direct and indirect antiox- regard, Young et al. [73] have reported that overexpression
idant effects by scavenging ROS and increasing Nrf2 activity, of both enzymes glyoxalase 1 and transglutaminase 2, an
followed by the induction of its target antioxidant genes enzyme catalysing polyamine conjugation/deamidation, led
[57]. The natural polyphenols are also substrates for ABC to increased tumour cell survival, drug resistance, and metas-
transporters as they bind to the active sites of the transporters tasis [74]. The use of photodynamic anticancer therapy is
and reduce drug efflux [58]. The prooxidant capacity of some particularly attractive because of its specificity and selec-
polyphenols (quercetin, epigallocatechin gallate) allowed tivity [75]. Hypericin, a naphthodianthrone, is a promising
their identification as chemotherapeutic adjuvants since they photosensitizer, which is feasible for photodynamic therapy,
selectively enhanced cytotoxic effects of chemotherapeutics for fluorescence diagnosis, and for topical applications [76].
[59]. Shin et al. have reported that some specific polyphenols Mikesova et al. [77] have shown that hypericin content in
triggered cell cycle arrest and apoptotic cell death in cisplatin- cells, GSH levels, and redox status correlated with hypericin-
resistant A2780/Cis human ovarian cancer cells [60]. induced photocytotoxicity. In contrast, resveratrol attenuated
cisplatin toxicity by maintaining GSH levels [78, 79]. It
3.1. Antioxidant-Associated Modification of Drug Transport- has also been demonstrated that buthionine sulfoximine,
ing Systems. Elevated GSH levels trigger chemoresistance an inhibitor of GSH biosynthesis, increased the sensitivity
by different pathways: direct interaction with drugs and of the cells to chemotherapeutics, while N-acetyl cysteine
ROS, prevention of protein and DNA damage, and induc- exhibited the reverse effect, particularly in drug-resistant cells
tion of DNA repair. For example, MRP1 causes efflux of [61, 62, 80]. Malabaricone-A, a diarylnonanoid with a potency
some xenobiotics (e.g., vincristine, daunorubicin) through of MDR reversal, induced depletion of GSH, inhibited
a cotransport mechanism with GSH [26, 6163]. Oxidative GPx activity, and caused redox imbalance [81]. Collectively,
stress was more cytotoxic towards B16 melanoma cells with molecular pathways-targets for MDR modulation by GSH
low GSH concentrations [64]. Tumour cells overexpressing controlling agents are schematically presented in Figure 3.
-glutamyl-transpeptidase were more resistant to H2 O2 and
chemotherapeutics, such as doxorubicin, cisplatin, and 5- 3.1.2. Overexpression of ABC Transporters: Effects of NADPH-
fluorouracil [65]. GST-related chemoresistance modulated Oxidase and CYP Inhibitors. NADPH-oxidase (NOX) is an
protein-protein interactions with members of the mitogen- oxidoreductase and plays crucial roles in cell growth, pro-
activated protein (MAP) kinases including c-Jun N-terminal liferation, and regulation of phosphatases and transcription
kinase 1 and apoptosis signal-regulating kinase 1 and altered factors via redox-sensitive cysteine residues [82]. Elevated
balance of kinases during drug treatment [66]. This complex NOX expression has been shown in breast cancer [83],
mechanism involved the interaction of promoter regions for colon cancer [84], and neuroblastoma cells [85]. Barth et al.
GST and GGT with NF-B and Nrf2 followed by upregulation have demonstrated that pharmacological block of glucosylce-
of several detoxification genes, such as ferritin, GSH-S- ramide synthase, a stimulator of NOX activity, substantially
reductase, and hemeoxygenase-1. Hypoxia induced breast improved cytotoxicity of chemotherapeutics in glioblastoma
cancer resistance protein (BCRP) expression in tissues by cells [86]. The molecular mechanism of anticancer effects of
interacting with heme and porphyrins thus increasing levels cisplatin involves activation of Akt/mTOR pathway regulated
of cytoprotective protoporphyrins [67]. Overexpression of by NOX-generated ROS, and NOX inhibition by diphenyl
BCRP is known to induce resistance to various chemother- iodonium was critical for cisplatin cytotoxicity [87].
apeutic drugs, such as topotecan and methotrexate [68].
Overexpression of the drug and xenobiotic metabolising
3.1.1. MDR Induction and Possibility to Inhibit It by Redox- cytochrome P450 enzymes for a long time has been con-
Active Substances Affecting Glutathione Metabolism. Definite sidered as one of the major mechanisms of chemoresistance
redox-active compounds, such as quinones, polyphenols, in solid tumours [88, 89]. Types 1 and 2 CYPs have been
oligomeric proanthocyanidins, ergothioneine, ovothiols, tan- proven to activate procarcinogens into ultimate carcinogens
nins, or terpenes, behave as redox modulators and trigger [90]. CYPs have become therapeutic targets in anticancer
redox-related events, such as ROS increase and GSH deple- protocols amid their involvement in the activation and/or
tion, causing apoptosis of cancer cells [69]. In general, redox inactivation of chemotherapeutic drugs [91]. Molina-Ortiz
modulations in cancer cells could initiate cell differentiation et al. reported that altered CYP expression played a crucial
or could induce apoptosis [70]. Acetaminophen, a widely role in the therapy of Rhabdomyosarcoma patients [92]. The
used drug to combat pregnancy-connected toxicity [71], in vitro antitumour action of natural product austocystin D
NADP+
NADP+
NADPHNADPH
NADPH NADPH TrxR
CAT
Trx red
GR Trx ox
HOOH
GSH Prxred
GSSG
GPx LOOH
HOOH Prxox
LOOH H2 O HOOH
Figure 3: Antioxidant and prooxidant systems as molecular targets for redox-active MDR modulators. Glutathione and enzymes involved in
the glutathione metabolism such as glutathione reductase (GR) and glutathione peroxidase (Gpx) as well as gamma-glutamyl cysteine ligase,
catalase, NADPH-oxidase, thioredoxin (Trx), and peroxiredoxins (Prx) are potential molecular targets for future MDR modulators.
has been explained by selective activation of CYP enzymes factor NF-B and promoting IL-6 synthesis, thus favouring
leading to DNA damage [93]. tumorigenesis and MDR [102].
3.2. Chemotherapy-Induced Inflammatory Responses May 3.2.1. Activation of NF-B-Dependent Pathways and Their
Cause Redox-Regulated Multidrug Chemoresistance. To Inhibition by Antioxidants. NF-B is the key transcription
increase clinical efficacy of chemotherapy and combat MDR, factor involved in the inflammatory pathway. NF-B is
molecular and cellular processes promoting inflammation constitutively active in many of the signalling pathways
have been targeted due to the common knowledge that implicated in cancer. Hyperactivation of NF-B in can-
(i) inflammatory cells are present within tumours and (ii) cer cells promotes cancer cell survival by inducing the
tumours arise at sites of chronic inflammation [94, 95]. upregulation of antiapoptotic proteins such as MnSOD and
Cancer promotion and progression stages are accelerated by Bcl-2 family members and the inhibition of proapoptotic
ROS generated by immune cells, mediators of inflammation proteins and is linked directly to the inflammation-induced
[96]. The induction of antioxidant defence enzymes in chemoresistance. NF-B protects against oxidative stress and
tumours as an adaptation to oxidative attacks from host activates transcription factor c-myc, MMP gene expression,
immune cells might contribute to chemoresistance. Thus, and tumour angiogenesis and remodels extracellular matrix,
hydrogen peroxide-resistant thymic lymphoma cells with while NF-B inhibition blocks cell proliferation [95, 103106].
increased catalase and total SOD activities, altered GSSG/ NF-B is associated with aberrant growth, resistance to apop-
GSH redox potential, and oxidised NADP+ /NADPH pool tosis, and overexpression of the genes involved in cell cycle
exhibited resistance to conventional chemotherapeutics, promotion in cancer cells. In a recent study, it has been shown
such as cyclophosphamide, doxorubicin, vincristine, and that isorhamnetin, a metabolite of quercetin, enhanced
glucocorticoids [97]. Chemotherapeutic agents caused antitumour effects of chemotherapeutic drug capecitabine
release of ATP into the extracellular space as they induced through negative regulation NF-B [107]. Singh et al. have
tumour cell death [98]. Following accumulation of adenosine reported that tea polyphenols inhibited cisplatin enhanced
in tumours through CD39 and CD73, immune responses activity of NF-B [108]. FADD-like IL-1beta-converting
were suppressed [99]. Clayton et al. showed that exosome- enzyme inhibitory protein (FLIP) is a potent inhibitor of
expressing CD39 and CD73 suppressed T cells through caspase-8-mediated apoptosis involved in NF-B activation.
adenosine production [100]. Oxidative stress has putative Talbott et al. revealed that FLIP regulates NF-B through
impact on the activation and regulation of protein kinase protein S-nitrosylation, a key posttranslational mechanism
C (PKC) with redox-sensitive regions in both N-terminal controlling cell death and survival strategies [109]. Overex-
regulatory domain and C-terminal catalytic domain. Rimessi pression of cyclin D1 in cancer cells was reported in cisplatin
et al. [101] have demonstrated that PKC induced resistance to chemoresistance. In contrast, reduction of cyclin D1 expres-
apoptotic agents following its translocation into the nucleus sion resulted in the increased sensitivity to cisplatin due to
as a result of oxidative stress. Nuclear PKC inhibitor restored reduced NF-B activity and apoptosis [110]. It was shown that
the apoptotic susceptibility of doxorubicin-resistant cells by vitamin E compounds, such as - and -tocotrienol, inhibited
forming a complex with the proinflammatory transcription NF-B activity, cell growth, cell survival, and tumour growth.
In parallel, -tocotrienol augmented sensitivity of pancreatic activation of TLR4 pathway [127]. The authors suggested that
cancer to gemcitabine [111]. simultaneous TLR4 block could reverse MDR to paclitaxel
Curcumin, a nontoxic food additive extensively used for and improve efficacy of the anticancer therapy.
food flavouring [112], has been found to suppress human Activation of TLRs in cancer cells seems to contribute
hepatoma through inhibition of tumour cell proliferation, cell to the tumour growth, cancer cell survival, and MDR via a
cycle arrest in G2/M phase, and induction of apoptosis [113]. signalling cascade involving cytokine/chemokine production
Numerous publications have reported curcumin as a sensi- [128]. It was shown that ligation to the TLR2 in lung cancer
tiser for a number of anticancer drugs [114], first of all, cis- cells induced activation of mitogen-activated protein kinases
platin [115]. Results of recent studies have also suggested that (MAPK) and NF-B, a classical pathway of survival strategy
curcumin was a reversal of induced MDR by multiple mech- [129]. Stimulation of TLR7/TLR8 in pancreas cancer cells
anisms such as the inhibition of ABC transporter expression resulted in elevated NF-B and COX-2 expression, increased
and function, activation of ATPase, and modulation of NF- cancer cell proliferation, and reduced chemosensitivity [130].
B activity during anticancer therapy [116]. In contrast, Active TLR-4/MyD88 signalling was also found in epithelial
caffeic acid, a natural phenolic, prevented antiproliferative ovarian cancer cells and influenced the drug response [131].
and proapoptotic effects induced by paclitaxel in lung cancer The relationships between the expressions of TLR-4, MyD88,
cells by the activation of NF-B-survivin-Bcl-2 axis, thus and NF-B have been examined in epithelial ovarian cancer
contributing to acquired MDR [117]. patients. Increased MyD88 expression was found to be
associated with poor survival rate [132].
3.2.2. Activation of Phosphoinositol-3 Pathway and Its Inhibi-
tion in a Redox Fashion. The phosphatidylinositol-3 kinase
3.3. Chemotherapy-Induced Redox-Dependent Stress Re-
(PI3K) pathway has been widely considered to be associated
sponses Leading to Adaptation. Along with killing cancer
with oncogenesis, cancer progression, and multiple hallmarks
cells, chemotherapeutic agents induce their stress and
of malignancy [118]. Consistently, PI3K pathway is a common
adaptive responses. Signalling pathways and gene expression
mechanism of resistance to antineoplastic agents [119]. Of
in response to chemotherapeutics play pivotal roles in the
note, resistance to PI3K inhibitors may also develop due to
development of acquired MDR [133]. Key functional aspects
aberrant compensatory signalling through other pathways
of cellular stress response include damage to membrane
[120]. The three main molecules in this pathway are PI3K,
lipids, proteins, and DNA and alterations in the redox status,
Akt, and mammalian target of rapamycin (mTOR). Recently,
energy metabolism, cell cycle, and proliferation [134]. Thus,
it has been reported that PI3K-mTOR inhibitor enhanced the
there is clear-cut evidence that upregulation of nonenzymatic
cytotoxicity of temozolomide, an advanced chemotherapy for
and enzymatic antioxidant defence, molecular chaperones,
malignant gliomas [121].
and stress responsive proteins are responsible for acquired
Since activation of integrins, proteins expressed on the
MDR [135]. These molecular pathways are potential targets
cytoplasmic membrane of malignant cells, is controlled
to enhance the cytotoxic effects of chemotherapeutics and to
directly by a redox site by disulfide exchange in their extracel-
overcome drug resistance.
lular domain, redox modifications of thiols could alter essen-
tial functions of integrins [122]. It was suggested that inactiva-
tion of VLA-4 integrin by nontoxic tellurium compound was 3.3.1. Nrf2 as a Perspective Target to Overcome MDR in
due to its binding to the thiol groups of cysteines that Tumours. Nrf2, a redox-sensitive transcription factor, plays
decreased PI3K/Akt/Bcl-2 signalling while enhancing drug a crucial role in redox homeostasis during oxidative stress.
sensitivity [123]. Gao et al. demonstrated that the natu- Nrf2 is sequestered in cytosol by an inhibitory protein Keap1
ral bioflavonoid apigenin reversed drug-resistant pheno- causing its proteosomal degradation [136]. In response to
type by its suppressor effect on PI3K/Akt/Nrf2 pathway in oxidative stress, Nrf2 translocates to nucleus and binds to
doxorubicin-resistant Nrf2 overexpressing cells [124]. ARE that increases the expression of antioxidant genes such
as hemeoxygenase-1, NAD(P)H: quinone oxidoreductase 1,
3.2.3. Activation of Toll-Like Receptors (TLRs) by Chemother- aldo-keto reductases, and several ATP-dependent drug efflux
apeutics and Inhibitory Effects of Redox Modulators. Recent pumps [137]. Many genes involved in phase II metabolism
studies implicate bacterial, parasitic, and viral infections as a are also induced by Nrf2, including GSTs, UGT, and UDP-
possible link between inflammation and carcinogenesis [125]. glucuronic acid synthesis enzymes [138]. While Nrf2 upregu-
One possible redox-sensitive signalling pathway connect- lation causes chemoresistance, its blockade sensitises a variety
ing infection-associated inflammation and carcinogenesis is of cancer cells, including neuroblastoma, breast, ovarian,
mediated by Toll-like receptors (TLRs). The hypothesis is that prostate, lung, and pancreatic cancer cells, to chemothera-
bacterial products, such as lipopolysaccharide, could activate peutic drugs [139]. Several flavonoid compounds have been
TLR4-MyD88 axis in tumour cells followed by the production reported to be potent Nrf2 inhibitors, such as epigallocat-
of proinflammatory cytokines, overexpression of antiapop- echin 3-gallate, luteolin, and brusatol [140, 141]. Nrf2 was
totic signals (XIAP and pAkt), and, finally, acquisition of upregulated in hepatocellular carcinoma and positive corre-
chemoresistance by ovarian cancer cells [126]. Both in vivo lation was found between Nrf2 expression and antiapoptotic
and in vitro experiments have shown that anticancer drug Bcl-xL and MMP-9 [142]. Quercetin treatment increased
paclitaxel exerted two opposite modes of action: killing of the total cellular amount and nuclear accumulation of Nrf2
breast cancer cells and enhancement of their survival through protein in malignant mesothelioma cells [143]. In vitro
suppression of Keap1 in human prostate and non-small- reactions and it has a high intracellular concentration. It
cell lung carcinoma cell lines elevated Nrf2 activity and serves as the major reducing peptide within all of the cells,
increased sensitisation to various chemotherapeutic agents due to its sulfhydryl group buffering and removing free radi-
and radiotherapy [144, 145]. These results demonstrated that cals generated during metabolic processes such as respiration.
Nrf2 inhibitors are effective adjuvants of chemotherapeutic GSH is an important factor for Bcl-2 ability to suppress
drugs. apoptosis. Chaiswing et al. observed that thiol redox status
and activities and expression of several antioxidant enzymes
3.4. Chemotherapy-Induced Prosurvival and Antiapoptotic exhibited distinct patterns in two prostate cancer cell lines
Cellular Strategies: Roles for Anti- and Prooxidants. Cellular at different growth phases, suggesting that modulation of
redox homeostasis is maintained by the balance between thiol redox status might be useful as a therapeutic tool to
endogenous antioxidant defence system, including antiox- modify cancer cell proliferation and tumour aggressiveness
idant enzymes such as SOD, catalase (CAT), GPX, GSH, [154]. Mitochondrial metabolism is altered in malignant cells
proteins, and low-molecular-weight scavengers, such as uric so that, in contrast to normal or benign cells, malignant
acid, coenzyme Q, and lipoic acid, and the prooxidant cells accumulate citrate due to low activity of mitochondrial
molecules, leading to the formation of several highly oxidis- aconitase, become citrate-oxidising cells, and exhibit low
ing derivatives. amounts of citrate. The higher rate of mitochondrial substrate
metabolism explains the increased levels of ROS associated
3.4.1. p53 Proapoptotic Protein. p53 is considered as the with malignancy and metastasis. The foregoing discussion
guardian of the genome, and several gene mutations encoding illustrates the importance of GSH in mitochondrial function
p53 have been detected in several tumour cells. Under and redox status, in determining the metastatic aggressive-
physiological conditions, activated p53 plays a key role in ness and sensitivity of cancer cells to chemotherapeutic
tumour prevention by promoting synthesis of antioxidant agents, and provides the rationale for mitochondrial GSH as
enzymes. ROS-induced DNA damage activates p53, leading a potential therapeutic target in cancer [155].
to apoptosis via the mitochondrial intrinsic pathway and
increasing the synthesis of prooxidant enzymes. Since p53 is 3.5. Chemotherapy-Induced Transcription and Activity of Aro-
a redox-sensitive factor, ROS negatively modulates its activity matic Hydrocarbons (Ah) Receptor. AhR is a basic helix-loop-
via oxidative modification of the cysteine residues at the helix transcription factor that, prior to ligand binding, is
DNA-binding site. It has been proposed that the loss of p53 stabilised in the cytoplasm by direct interaction with several
function in cancer cells is associated with their ability to avoid proteins such as heat shock protein 90, its cochaperon, and
apoptosis [146]. Mutations of p53 are involved in resistance to X-associated protein 2. Upon binding to aromatic ligands,
chemotherapy [147]. It was demonstrated that p53 regulated toxins, drugs, phytochemicals, and sterols, the AhR-ligand
Nrf2 negatively and interfered with the ability of Nrf2 to bind complex shuttles from cytoplasm to the nucleus, where it
to DNA. A low level of p53 favoured binding of Nrf2 to DNA. heteromerises with the AhR nuclear translocator (Arnt) to
In a recent study, the treatment with bortezomib, which is form the transcription complex able to bind to xenobiotic
a selective proteasome inhibitor, induced Myelocytomatosis responsive elements (XRE) DNA-binding motifs located in
Viral Oncogene Neuroblastoma (MYCN) downregulated p53 the promoter region of the target drug metabolising genes,
expression, leading to cell survival in neuroblastoma [148]. such as phase I (mainly, Cyp1 subfamily) and II metabolising
enzymes and Nrf2 [156158]. One of the downstream targets
3.4.2. Bcl-2 Antiapoptotic Protein. Bcl-2 protein is a member for AhR is BCRP encoded by ABCC3 gene [159161]. The
of a family of apoptosis-modulating proteins which pro- coordinate AhR- and Nrf2-dependent transcriptional regula-
tects against a variety of apoptotic stimuli, mainly acting tion of human UGTs by utilising both XRE- and ARE-binding
at the mitochondrial level. In addition to its antiapoptotic motifs takes place to protect cells from xenobiotic and oxida-
action, Bcl-2 has been shown to exert potent antioxidant tive stresses [162]. The elegant study with genetically modified
effects [149] such as protection of lipid membranes against mice has clearly demonstrated that Nrf2 is required for
peroxidation reactions, maintenance of cellular redox status ligand-associated induction of classical AhR battery genes
(i.e., NADH/NAD+ and GSH/GSSG in a reduced state) in NQO1, GST isoforms, and UGTs [163]. Apart from metabolic
response to oxidative stress [150], and elevation of cellular enzymes, a number of growth factors, cytokines, chemokines,
levels of glutathione and reducing equivalents [151]. Bcl-2 and their receptors are downstream gene targets for activated
upregulates antioxidant defence systems, and its mitochon- AhR [164, 165]. AhR is also functionally connected with epi-
drial localisation contributes to achieving this effect. Bcl-2 is dermal growth factor receptor, presumably, through NF-B-
capable of forming ion channels as a regulator of mitochon- regulated pathway [166], thus influencing the epithelial cell
drial permeability transition [152]. Mitochondrial permeabil- proliferation. AhR can also cross-talk and directly interact
ity transition blockade prevents release of cytochrome c, an with proteins involved in major redox-regulated signalling
apoptosis initiating factor. Herrmann et al. reported that Bcl- pathways such as NF-B and various kinases such as Src,
2 selectively regulates nuclear localisation of cell death reg- JNK, p38, and MAPK [167] and with oestrogen receptors
ulators such as p53 and NF-B [153]. Cellular redox state via to mediate oestrogen metabolism [168, 169]. Recent studies
thiols plays a major role in regulating mitochondria-mediated have unraveled unsuspected physiological roles and novel
events during apoptosis, and oxidation of mitochondrial alternative ligand-specific pathways for this receptor that
thiols is an apoptotic sensor. GSH is involved in detoxifying allowed hypothesising numerous pharmacological roles of
AhR ligands useful for the development of a new generation UV- or FICZ-upregulated AhR-Cyp1A1-Cyp1B1 axis in
of anti-inflammatory and anticancer drugs [159, 170]. human keratinocytes [184], suggesting their potency as
The AhR-mediated regulation of aromatic hydrocarbons topical MDR suppressors/reversals.
metabolism has been implicated in a variety of cancers [164,
171] affecting different stages of carcinogenesis. If metabolic 4. Conclusions
activation of the organic molecules increased the levels of
their adducts with DNA thus promoting cancer initiation, The multiple pleiotropic interactions of redox-active
anticancer drug- or toxin-induced AhR activation played molecules so far demonstrated on the molecular pathways
a pivotal role in cancer promotion and progression [172, controlling cellular MDR, from xenobiotic cellular uptake
173]. Elevated AhR expression associated with constitutive inhibition to the modulation of phase I/II enzyme
nonligand activation has been found in several cancers as detoxification, to the inhibition of Toll-like receptor
evidenced by the nuclear localisation of AhR and induced activation and/or AhR expression and function, provide a
downstream gene Cyp1A1 [174, 175]. Stable knockdown of consistent rationale for the necessity of more intense and
AhR decreased the tumorigenic and metastatic properties of systematic research efforts in the field of anti-/prooxidant
breast cancer cell line in vitro and in vivo. On the other hand, adjuvants for anticancer chemotherapy, to attempt clinically
AhR overexpression in nontumour human mammary epithe- effective MDR inhibition with tolerable toxicity.
lial cells transformed them in cells with malignant phenotype The design and implementation of more selected and
[176]. Of importance, AhR knockdown downregulated the targeted clinical studies centred on redox-active candidate
expression of ABCC3; overexpression of this gene in breast MDR-interfering molecules will possibly contribute to over-
cancer has been strongly associated with acquired MDR coming the presently dominating clinical practice, which
[177] and resistance to paclitaxel, a drug widely used in the confers a constantly growing interest in redox modulators
treatment of metastatic breast cancer [178]. Inherited poly- and antioxidants as a mere palliative against the potent
morphisms in AhR, for example, substitution Arg554Lys, and cytotoxicity of conventional and biologic anticancer drugs.
its machinery [179, 180] or presence of endogenous ligands-
stimulators for the receptor (cAMP, bilirubin, prostaglandins,
oxidative lipids, etc.) could be implicated into inherited
Abbreviations
MDR. ABC: ATP-binding cassette
To suppress AhR transcription pharmacologically several AhR: Aryl hydrocarbon receptor
approaches have been proposed, including the modulation Akt: Protein kinase B
of protein-protein interaction between transcription factor, AP-1: Activator protein 1
coactivators, and corepressors [160, 167, 181]. A number of ARE: Antioxidant response element
dietary polyphenols with redox properties (resveratrol, quer- Bcl2: Apoptosis regulator Bcl-2
cetin, curcumin, etc.), indoles, tryptophane metabolites, BCRP: Breast cancer resistance protein
bilirubin, and oxidised products of lipid metabolism have CAT: Catalase
been suggested as nontoxic ligands-activators or ligands- COMT: Catechol-O-methyl transferase
inhibitors of AhR expression by competitive and noncom- CYP: Cytochrome P450
petitive pathways [181, 182]. If ligands-activators of AhR are EM: Extensive Metabolisers
regarded as potential anti-inflammatory agents [181184], FLIP: FADD-like IL-1beta-converting enzyme
redox-active ligands able to suppress AhR expression/ inhibitory protein
functions could be candidates for MDR reversals. For GPx: Glutathione peroxidase
example, 7-ketocholesterol, a major dietary oxysterol, may GSH/GSSG: Reduced glutathione/oxidised glutathione
actually strongly inhibit AhR activation [185]. Alpha- GST: Glutathione-S-transferase
naphthoflavone is considered as a classical AhR antagonist JNK1: c-Jun N-terminal kinase 1
blocking activation of XRE-containing reporter gene and Keap 1: Kelch-like ECH-associated protein 1
Cyp1 upregulation in hepatoma cells [186]. However, alpha- MDR: Multiple drug resistance
naphthoflavone is also a partial agonist of AhR and acts as a MMP: Matrix metalloproteinase
competitive inhibitor exclusively in the presence of another mTOR: Mammalian target of rapamycin
agonist. Recently, more selective pure ligands-antagonists of MXR: Multiple xenobiotic resistance
AhR have been developed such as 2-methyl-2H-pyrazole-3- NADPH: Nicotinamide dinucleotide phosphate,
carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide, 3 - reduced form
methoxy-4 -nitroflavone, 3 ,4 -dimethoxyflavone, and 6,2 , NAT: N-Acetyl transferase
4 -trimethoxyflavone. These were able to block the induction NF-B: Nuclear factor kappa B
of Cyp1A1-dependent ethoxyresorufin O-deethylase (EROD) NQO1: NAD(P)H: quinone oxidoreductase 1
activity [187190]. They all belong to redox-active flavones NOX: NADPH-oxidase
and after proper clinical studies on safety and efficacy could Nrf1/2: Nuclear factor E2-related factor 1/2
be feasible for combinatory anticancer therapy to combat P-gp: P-glycoprotein
MDR. Among a number of plant polyphenols used for topical PPI3K: Phosphoinositol-3-kinase
application, exclusively the phenylpropanoid verbascoside PKC: Protein kinase C
and flavonoid quercetin proved to be strong inhibitors of PM: Poor Metabolisers
Prx: Peroxiredoxins cytochrome P450 super-family in human extrahepatic tissues,

RNS: Reactive nitrogen species Current Drug Metabolism, vol. 9, no. 2, pp. 129143, 2008.
ROS: Reactive oxygen species [12] D. Kluth, A. Banning, I. Paur, R. Blomhoff, and R. Brigelius-
SNP: Single nuclear polymorphism Flohe, Modulation of pregnane X receptor- and electrophile
SOD: Superoxide dismutase responsive element-mediated gene expression by dietary
polyphenolic compounds, Free Radical Biology and Medicine,
STAT3: Signal transducer and activator of
vol. 42, no. 3, pp. 315325, 2007.
transcription 3
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Conflict of Interests
[16] A. K. Jaiswal, Nrf2 signaling in coordinated activation of
The authors declare that they have no conflict of interests. antioxidant gene expression, Free Radical Biology and Medicine,
vol. 36, no. 10, pp. 11991207, 2004.
[17] B. Fadeel and S. Orrenius, Apoptosis: a basic biological phe-
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http://dx.doi.org/10.1155/2016/7909380
Review Article
Is Modulation of Oxidative Stress an Answer? The State of the
Art of Redox Therapeutic Actions in Neurodegenerative Diseases
Valerio Chiurchi,1,2 Antonio Orlacchio,3,4 and Mauro Maccarrone1,2

1
School of Medicine and Center of Integrated Research, Campus Bio-Medico University of Rome, Rome, Italy
2
European Center for Brain Research (CERC), Laboratory of Neurochemistry of Lipids, IRCCS Santa Lucia Foundation, Rome, Italy
3
European Center for Brain Research (CERC), Laboratory of Neurogenetics, IRCCS Santa Lucia Foundation, Rome, Italy
4
Department of System Medicine, University of Rome Tor Vergata, Rome, Italy
Correspondence should be addressed to Valerio Chiurchiu; v.chiurchiu@hsantalucia.it
Received 12 August 2015; Accepted 18 October 2015
Academic Editor: Liudmila Korkina
Copyright 2016 Valerio Chiurchiu et al. This is an open access article distributed under the Creative Commons Attribution
cited.
The central nervous system is particularly sensitive to oxidative stress due to many reasons, including its high oxygen consumption
even under basal conditions, high production of reactive oxygen and nitrogen species from specific neurochemical reactions, and
the increased deposition of metal ions in the brain with aging. For this reason, along with inflammation, oxidative stress seems
to be one of the main inducers of neurodegeneration, causing excitotoxicity, neuronal loss, and axonal damage, ultimately being
now considered a key element in the onset and progression of several neurodegenerative diseases, including Alzheimers disease,
Parkinsons disease, amyotrophic lateral sclerosis, multiple sclerosis, and hereditary spastic paraplegia. Thus, the present paper
reviews the role of oxidative stress and of its mechanistic insights underlying the pathogenesis of these neurodegenerative diseases,
with particular focus on current studies on its modulation as a potential and promising therapeutic strategy.
1. The Role of Oxidative Stress in chain [6] and through NADPH oxidase, an enzyme that uses

Neurodegeneration NADPH to reduce O2 , thus generating large amounts of O2
on the membrane surface as a toxic agent during elimination
Although molecular oxygen (O2 ) is crucial for life of most of pathogens [7]. The reactivity of NO with ROS leads to
organisms, it is not totally innocuous. The deleterious effects the formation of many other reactive species, termed reac-
of O2 are thought to result from its univalent metabolic reductive nitrogen species (RNS), which include the key effector
tion that leads to the formation of chemically reactive and molecule ONOO , but also other species such as nitrogen
toxic species, known as reactive oxygen species (ROS). These dioxide (NO2 ), dinitrogen trioxide (N2 O3 ), and dinitrogen
include molecules that contain oxygen-centered free radicals,

tetroxide (N2 O4 ) [8]. In mammals, NO is an essential
such as the superoxide radical anion ( O2 ), the hydroxyl biological molecule mostly generated by a family of specific
radical (HO ), hydroperoxyl radical (HO2 ), and peroxyl NO synthase (NOS) isozymes: endothelial (eNOS), neuronal
radicals (ROO ), as well as nonradical derivatives of O2 like (nNOS), and inducible (iNOS) isozymes [9]. However, NO
hydrogen peroxide (H2 O2 ), hypochlorous acid (HOCl), and can be produced also by other redox enzymes such as xan-
peroxynitrite (ONOO ) [14]. Several sources, either exoge- thine oxidase or nonenzymatically by guanidine-substitute
nous or endogenous, contribute to intracellular ROS for- L-arginine analogs in the presence of NADPH [10, 11]. Not
mation. Exogenous sources include radiation, atmospheric surprisingly, the major producers of ROS and RNS are indeed
pollutants, and chemicals [5]. Endogenously, ROS originate immune cells and specifically phagocytic cells, either resident

mainly from mitochondria, when O2 is formed by electrons cells in the brain (i.e., microglia) or infiltrated leukocytes, due
leaking between complexes I and III of the electron-transport to their elevated expression of NADPH oxidase, iNOS, and
xanthine oxidase [12, 13]. However, cells are equipped with causes of such alteration of oxidative metabolism. Along with
enzymatic and nonenzymatic antioxidant systems to elim- strong and persistent production of these reactive species,
inate ROS and RNS, thus maintaining redox homeostasis. also associated with significant upregulation of their pro-
Antioxidants include naturally occurring molecules of high ducing enzymes (myeloperoxidase, xanthine, and NADPH
or low molecular weight, as well as nutritional antioxidants, oxidases), increases in lipid and DNA oxidation products
whose action is strictly linked to their bioavailability. Nat- (i.e., OH8dG, 8-hydroxydeoxyguanosine) have been also
urally occurring antioxidants are mainly enzymes such as reported. Free radicals can also activate certain transcrip-
superoxide dismutase (SOD), catalase, glutathione peroxi- tion factors, like NF-B, which upregulate the expression
dase/reductase (GPx/GR), and peroxiredoxin or molecules of many genes involved in neurodegenerative diseases,
like glutathione (GSH), uric acid, pyruvate, amino acids, including proinflammatory cytokines and vascular adhesion
transferrin, ferritin, and caeruloplasmin. On the other hand, molecules. Additionally, redox reactions are involved in the
nutritional antioxidants include lipid-soluble antioxidants activity of matrix metalloproteinases (MMPs), which are
(-tocopherol, carotenoids, quinones, and some polyphe- important to cell trafficking into the CNS [17]. Interestingly,
nols) and water-soluble antioxidants (ascorbic acid and some along with increased ROS and RNS, direct examination
other polyphenols) [2]. Oxidative stress is strictly dependent of brain tissues from patients affected by neurodegenera-
on the balance between the rate of radicals production and tive diseases also revealed a weakened cellular antioxidant
that of their clearance. This overall balance seems to be defense, especially due to impairment and/or decrease of
under the regulation of transcription factor nuclear factor- relevant antioxidants such as superoxide dismutase, catalase,
E2-related factor (Nrf2), which is indeed a central component glutathione/glutathione peroxidase, -tocopherol, and uric
of cellular defense against oxidative stress [14]. Intriguingly, a acid [18]. Indeed, lower levels of antioxidants may pro-
very recent discovery reported the presence of a specific promote increased activity of lipoxygenase (that catalyzes one
tein, termed negative regulator of ROS, that is, NRROS, which branch of the arachidonate cascade), thereby increasing the
is capable of regulating the production of ROS by modulat- immunoinflammatory processes within the brain. In line
ing their generation from phagocytes during inflammatory with this, excessive ROS can stimulate T-cell activity through
responses [15]. In particular, this regulator, which is local- the arachidonate cascade or can produce direct/indirect
ized in the endoplasmic reticulum, directly interacts with damage to the blood brain barrier (BBB) or to neurons [19].
the membrane-bound subunit gp91(phox) of the NADPH
oxidase complex and facilitates the degradation of NOX1 and 2. Alzheimers Disease
NOX2 proteins, thereby modulating ROS production.
The central nervous system (CNS) is particularly sensitive Alzheimers disease (AD) is probably the most common neu-
to oxidative stress and this is due to several reasons. One rea- rodegenerative disease, accounting for 60% to 70% of cases of
son is its high consumption of O2 (the brain can metabolize dementia with nearly 44 million affected people worldwide,
up to 4 1021 molecules of glucose per minute.) A second and although its etiology is still unclear, it is characterized
reason is the high production of ROS and RNS, which orig- by the presence of brain amyloid plaques and neurofibrillary
inate from specific neurochemical reactions (e.g., dopamine tangles whose accumulation ultimately leads to extensive
oxidation), in addition to the sources discussed previously. neuronal loss and progressive decline of cognitive function [2,
A third reason is the increasing deposition of metal ions in 39, 40]. They are deposits of proteins distributed throughout
the brain with aging, catalyzing the production of increasing the brain of AD patients, particularly in the entorhinal cortex,
levels of ROS and RNS [2]. Another reason is the relatively hippocampus, and temporal, frontal, and inferior parietal
high abundance of lipids within the CNS (i.e., myelin), which lobes. Amyloid plaques are primarily composed of aggregates
are particularly sensitive to oxidation. For instance, HO2 is of -amyloid (A), as well as other protein aggregates
particularly relevant for the in vivo lipid peroxidation and (e.g., hyperphosphorylated Tau, ubiquitin, and presenilins
it acts via two different pathways: one that is lipid perox- 1 and 2), whereas neurofibrillary tangles are aggregates of
ides independent and the other one that is lipid peroxides hyperphosphorylated Tau protein [41]. The production of
dependent [4], leading to the formation of several secondary ROS and its involvement in AD pathogenesis are supported
breakdown products including epoxides and saturated and by the significant amount of lipid peroxidation detected in
unsaturated aldehydes such as malondialdehyde (MDA) and the brain of AD patients, as well as by the increased levels
4-hydroxynonenal (HNE), cyclopentenones (i.e., cyclopen- of HNE found postmortem in their cerebrospinal fluid (CSF)
tenone isoprostanes), and nitro-fatty acids (NO2 -FAs). Accu- [42]. Further, -amyloid-induced damage promotes the gen-
mulated evidence indicates that oxidative stress plays a eration of ROS, contributing to cell death and neurodegen-
major role in the pathogenesis of several neurodegenerative eration, and induces also glial recruitment and activation,
diseases, including Alzheimers disease (AD), amyotrophic thus triggering local inflammation. Further, oxidative stress
lateral sclerosis (ALS), Parkinsons disease (PD), multiple promotes abortive cell cycle reentry and hence apoptosis of
sclerosis (MS), and hereditary spastic paraplegia (HSP). High nerve cells of the adult brain and gene duplication without
levels of ROS and RNS are consistently generated by infil- cell division, leading to aneuploidy and DNA damage [39]. In
trating monocytes/macrophages and activated microglia and addition, oxidative stress can damage DNA, leading to strand
have been implicated as mediators of neurodegeneration and breaks and large deletions, and can affect various enzymatic
axonal damage typical of these disorders [16] (Figure 1). Mito- and mitogenic pathways. Interestingly, oxidative stress has
chondrial dysfunction in these cells is likely to be one of the been shown to decrease neurogenesis in the adult brain,
Oxidative stress

O2 H2 O2
HO
ROO
NO HO2
Inflammation Neurodegeneration
Monocytes/macrophages Astrocyte
Th-1/Th-17
T and B lymphocytes cytokines
Th-2 cytokines
Neuron
Oligodendrocyte
Dendritic cells
Figure 1: Cross talk between oxidative stress, inflammation, and neurodegeneration. The different reactive species are produced by several
cell types, either by resident brain cells (i.e., glia) or by infiltrated leukocytes (i.e., monocytes/macrophages and dendritic cells), and affect
both inflammatory processes, by enhancing cytokine release from proinflammatory T cells, and neurodegeneration, by inducing neuronal

cell death and axonal loss. O2 : superoxide radical anion; HO : hydroxyl radical; ROO : peroxyl radical; HO2 : hydroperoxyl radical; H2 O2 :

hydrogen peroxide; NO : nitric oxide; Th: T-helper.
thus limiting its neurodegenerative capacity [4345]. For the contributing factors that foster the clinical manifestations
treatment of AD, the current therapy involves drugs that are of this neurodegenerative disease should be unraveled, par-
only able to reduce symptoms or delay disease progression ticularly in relation to their effects on adult neurogenesis
such as acetylcholinesterase inhibitors and those targeting the and on synaptic communication. A more sophisticated redox
glutamatergic system. Concerning redox homeostasis, sev- approach involves the interaction between heavy metals and
eral antioxidant strategies are under study and aim not only at A. For instance, ionic zinc and copper are able to accelerate
reducing the deleterious activities of ROS, but also at promot- the aggregation of A and to promote its neurotoxic redox
ing the regenerative capacity of the adult brain [46] (Table 1). activity by induction of oxidative cross-linking of the pep-
These drugs have been experimented in rodent models of AD tide into stable oligomers [49]. Therefore, small molecules
and include garlic extracts, curcumin, melatonin, resveratrol, targeting these interactions are currently under clinical trials
Gingko biloba extracts, green tea, and vitamin C. Although and hold promise as disease-modifying agents for AD. These
the clinical value of these antioxidants for the prevention of novel drugs are referred to as metal protein attenuating
AD is often elusive, some of these compounds can be recom- compounds (MPAC) [50]. These are different from classical
mended based upon epidemiological evidence and already metal chelators, inasmuch as they bear a relatively low affinity
known benefits for prevention of other pathologies [47, 48]. for metals and are able to cross the BBB. Furthermore, MPAC
Yet, further long-term studies can be recommended to better stabilize metal homeostasis and interaction with proteins,
understand their mode of action. Vitamin E supplementation rather than binding and eliminating metals from tissues. For
in moderately severe AD is to date the most promising instance, clioquinol is a zinc/copper ionophore that facilitates
approach, although its efficacy is fairly limited and does not the clearance of A aggregates in the cortex of animal
apply to all AD patients. The beneficial effect of vitamin E is models of AD. Its ionophoric properties liberate copper and
mainly exerted against peroxidation of membrane lipids of zinc ions trapped within amyloid plaques, facilitating the
neurons. Several drugs with vitamin E are in use (e.g., Sursum, reuptake of these essential metal ions into cells and hence
Ephynal, and Rigentex) and are orally administered twice a promoting memory functions such as long-term potentiation
day. Yet, their therapeutic efficacy has not been thoroughly [20]. Oral treatment with this MPAC has been shown to
investigated and lack of data reveals the limitations of general have striking effects in transgenic mouse models of AD,
antioxidant therapies, whereby they simply lower oxidative markedly improving learning and memory within days,
stress rather than interfering with the molecular mechanisms paralleled by a significant reduction of A content [2]. Other
underlying disease pathogenesis [47]. In order to design MPAC molecules are being tested in preclinical murine
more effective antioxidant therapies against AD, the multiple models of AD with the aim of assessing their effect on
Table 1: Current redox clinical investigations for the treatment of GSH and increased levels of GSSG in the SNpc. This could
neurodegenerative diseases. be a critical primary event that weakens or abrogates the
natural antioxidant defense of the cell, thereby triggering
Redox therapy Clinical application degeneration of the nigral neurons and causing PD [51].
Alzheimers disease [20, 21] Since dysregulation of metal ion homeostasis is a potential
Vitamin E Parkinsons disease [22, 23] catalyst to further production of reactive species, the highly
Multiple sclerosis [24, 25] oxidative environment for DA interaction with -synuclein,
Parkinsons disease [22, 23] and the resulting oxidant-mediated toxicity and protein
Multiple sclerosis [24, 25] aggregation, is one of the most likely underlying mechanisms
Polyphenols
Hereditary spastic paraplegia for PD. Thus, the destruction of neuronal cells occurs
[ClinicalTrials.gov: NCT02314208] as a result of self-propagating reactions that involve DA,
Parkinsons disease [2629] -synuclein, and redox-active metals [52]. As for AD, also
Coenzyme Q10
Multiple sclerosis for PD no cures are available yet. However, pharmacological
MPAC Alzheimers disease [3032] treatment and surgery could help with symptom relief.
(i.e., clioquinol) Parkinsons disease [33, 34] The most commonly used drugs to treat motor symptoms
-3 PUFAs Multiple sclerosis [35, 36] are L-DOPA, a precursor of dopamine, which is usually
used in combination with a DOPA decarboxylase inhibitor
Metalloporphyrins Amyotrophic lateral sclerosis [37] and a catechol-O-methyltransferase inhibitor; dopamine
Pramipexole agonists; and monoamine oxidase inhibitors. These enzymes
Amyotrophic lateral sclerosis [38]
(i.e., KNS-760704) are all involved in the chemical inactivation of several
MPAC: metal protein attenuating compounds; PUFAs: polyunsaturated fatty neurotransmitters, including dopamine. In the early stages of
acids. the disease, the treatment aims at controlling both symptoms
and side effects caused by dopaminergic enhanced activity,
but when the disease gets more severe surgery can be useful.
memory loss and evaluating therapeutic efficacy and toxicity However, in the last stages of PD, palliative care seems to be
in vivo. the only alternative to improve the quality of life [53]. Over
the last decade, neuroprotective approaches for PD have been
3. Parkinsons Disease tried with the aim of slowing the rate of disease progression
by decreasing oxidative stress (Table 1). There has been
Parkinsons disease (PD) is the second most common much interest in the use of supplemental vitamin E, which
neurodegenerative disorder, affecting an estimated 10 seems to inhibit cell death of neuronal cells of SNpc. Regular
million people worldwide, which produces muscular rigidity, consumption of vitamin E-rich foods may have the potential
bradykinesia, tremor of resting limbs, and loss of postural to decrease the risk or delay the onset of PD. Even -carotene
balance. The basic neuropathology of PD involves degen- seems to reduce the risk of PD onset, although no studies to
eration of pigmented neurons in substantia nigra, resulting prove its efficacy are available yet. On the other hand, intake
in depletion of striatal dopamine (DA) and its metabolites. of vitamin C and flavonoids did not show any significant
The pathological hallmarks of PD are large cytoplasmic beneficial effect in either prevention or treatment of PD.
inclusions called Lewy bodies, which occur predominantly Overall, high intake of dietary antioxidant supplements
in the melanin-containing neurons of substantia nigra pars (mainly vitamin E) might protect against the occurrence of
compacta (SNpc), and contain aggregates of -synuclein. PD rather than treating its symptoms [54, 55]. The use of
Another gene encoding a protein termed parkin is involved melatonin and -lipoic acid has also been investigated for
in autosomal recessive Parkinsonism. Parkin is one member PD treatment, but their effects, though promising, have not
of the family of ubiquitin ligases and may be involved in been fully characterized [56]. The antioxidant with the most
normal turnover of -synuclein. Although the exact cause efficacious therapeutic potential is coenzyme Q10. Indeed,
of PD is still obscure, both environmental and genetic several clinical trials based on coenzyme Q10 have been
factors have been implicated in its pathogenesis [21]. Recent undertaken, showing significant beneficial effects on motor
evidence points toward a putative role of mitochondrial functions [22, 23, 57]. Currently, a phase III clinical trial is
dysfunction and oxidative stress as well as prooxidant ongoing and is based on combinations of coenzyme Q10
environmental toxicants in the pathogenesis of PD [30, 31], and vitamin E or creatine, with the aim of evaluating the
as demonstrated in postmortem brains from PD patients. effective dosage according to the disease stage. Initial results
Apparently, there is a specific chemical fingerprint indicative seem to document additive neuroprotective effects in terms
of the damaging oxidative events, that is, higher levels of of significant reduction of DA depletion in the striatum and
cholesterol hydroperoxide, MDA, HNE, and OH8dG. One loss of tyrosine hydroxylase neurons in the SNpc, as well as
of the suggested causes of oxidative stress in the SNpc is reduction in lipid peroxidation and pathologic accumulation
the production of ROS during normal DA metabolism. of -synuclein in the same SNpc neurons [26]. Further, a
In human SNpc, the oxidation products of DA (mainly 6- double blind phase I/IIa clinical study proved the safety and
hydroxydopamine) may polymerize to form neuromelanin, tolerability of intranasal GSH administration to early and
which may also be toxic by inducing apoptosis [32]. untreated patients, although pharmacokinetic and dose-
Furthermore, postmortem studies revealed reduced levels of finding investigations still need to be verified [27]. As already
described for AD, another potential therapeutic strategy lies definitive cure for ALS, the Food and Drug Administration
in the modulation of heavy metals, whereby the control of (FDA) has already approved the first molecule to treat the
their bioavailability could prevent not only the increase of disease, that is, riluzole. This drug is thought to reduce the
oxidative stress through metalloredox reactions, but also motoneuron-associated damage by affecting the release of
their interaction with other proteins like -synuclein. Thus, glutamate. Treatment with riluzole of ALS patients in clinical
MPAC have been also tested in PD [28] and clioquinol trials only elicited a three-month improvement of survival
has been reported to reduce cell death of substantia nigra rate, with the subjects needing constant monitoring of liver
neurons by 50% [29]. Of note, the inhibition of monoamine damage and other side effects [64]. Nonetheless, this first
oxidase isoforms reduces the formation of dopamine-derived attempt of a specifically aimed therapy nurtures the hope
peroxides and the subsequent generation of reactive species that the clinical course of ALS might be managed by new
and of the overall oxidative stress of substantia nigra. kinds of treatments or combinations of new drugs. Moreover,
several antioxidant molecules have been tested as putative
4. Amyotrophic Lateral Sclerosis therapeutic agents in the treatment of ALS (Table 1). Such
compounds include NAC and N-acetylmethionine (NAM),
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegener- vitamins C and E, resveratrol, dithiotreitol (or any of its iso-
ative disease characterized by the death of both upper and mers), and dithioeritrol. However, although such antioxidant
lower motor neurons of the brain, brain stem, and spinal cord, agents never caused noxious effects, all of them failed in
overall leading to progressive weakness and atrophy of skele- eliciting any kind of significant effect on patients survival rate
tal muscles [2, 33]. Approximately 10% of the cases are inher- [65]. To date, two antioxidant compounds have been used in
ited in an autosomal dominant manner, and 1/5 of these famil- clinical trials. The first one is manganese-metalloporphyrin,
ial ALS patients carries mutations in the Cu/Zn-SOD (SOD- which was able to extend survival rate in murine models [66].
1) gene, suggesting involvement of ROS in disease pathogen- Metalloporphyrins are compounds made by a tetrapyrrole
esis [34]. The toxicity of mutant SOD-1 seems to be due to ring that coordinates a central metal atom. Two phase I trials,
gain of function of this enzyme, whereby its catalytic activity conducted to assess possible drug-associated toxicity, showed
is enhanced with abnormal substrates like ONOO , thus sus- very good tolerance of this compound even at fairly high
taining nitration of tyrosine and subsequent oxidative stress. doses (up to 2 mg/kg/day), with ALS patients showing an
This may also be related to impaired ability of mutant SOD- excellent pharmacokinetic profile. The second antioxidant
1 to bind zinc, because in vivo the mutant enzyme is likely drug under investigation is KNS-760704, a pramipexole
to denature more quickly than the normal form, releasing enantiomer, usually used for treatment of PD patients, which
zinc and copper ions. Oxidative stress may also be involved acts as a dopamine receptor agonist [67]. This compound
in misfolding of mutant SOD-1, to yield abnormal protein possesses ROS-scavenging activity and has been proven to
aggregates that can be found as early as in 1-month-old SOD- extend the lifespan of ALS animal models. Moreover, a
1-mutant mice [58]. Also, the disorganization of intermediate recent phase II clinical trial also reported that KNS-76074
filaments could be due to mutant SOD-1-induced toxicity, is able to exert protective action against oxidative stress-
as these cytoskeletal proteins are vulnerable to oxidative associated neurotoxic damage, which led this compound to
damage [59]. In addition, protein carbonyl and nitrotyrosine be tested in phase III trials in order to evaluate its efficacy and
modifications, which are indexes of protein oxidation, were tolerability. New therapeutic approaches are currently being
found to be elevated in the majority of patients with sporadic developed with the aim of blocking the production of SOD-
ALS, suggesting that oxidative stress may indeed be involved 1 mutated forms, a strategy that could likely ameliorate the
in all types of ALS. Other mechanisms that have been clinical course of patients affected by the familial form of
implied in ALS, such as excitotoxicity and defective axonal ALS. Curiously, a recent study pointed out that the mutant
transport, may be consequences of oxidative stress [60]. SOD-1 transgenic mice model of ALS is not representative
What remains as yet unclear is whether this increased redox of the human sporadic form, accounting for most failures in
stress is a primary defect or a secondary consequence of the experimental or clinical research.
disease. An interesting study has demonstrated that SOD-1-
mutant ALS transgenic mice activate cellular Nox2 activity 5. Multiple Sclerosis

and subsequent O2 production in spinal cord microglia,
through disruption of the redox-sensitive regulation of Rac1- Multiple sclerosis (MS) is a chronic inflammatory, pro-
dependent Nox activation [61]. Of note, Nox enzymes control gressive, and degenerative disorder of autoimmune origin
relevant proinflammatory signaling pathways involved in the characterized by intermittent episodes of demyelination and
progression of ALS, such as those mediated by IL-1 and axonal loss or damage in the CNS. Although its etiology
TNF- via redox-dependent activation of NF-B. Finally, has not been fully elucidated yet, it is likely that both
marked elevation of 2-thiobarbituric reactive substances in genetic and environmental components play a crucial role in
plasma (i.e., MDA) was found both in mutant SOD-1 mice disease onset and progression, and it is now well recognized
and in patients with sporadic ALS [62, 63]. However, plasma that immunological mechanisms are the initial trigger [2,
concentrations of antioxidants like -tocopherol, -carotene, 16, 37]. MS is classified into four independent subtypes or
ubiquinol-10, and GSH, as well as SOD activity in red forms: relapsing-remitting (RR), primary progressive (PP),
blood cells, were not significantly different between ALS secondary progressive (SP), and progressive relapsing (PR);
patients and healthy subjects [63]. Even though we still lack a the former is the most prevalent form and accounts for
approximately 85% of all cases [38]. Interestingly, its patho- by the evidence that excessive ROS can stimulate T-cell
genesis and pathophysiology have been extensively studied, activity via the arachidonate cascade, or they can produce
especially on experimental autoimmune encephalomyelitis direct/indirect damage to the BBB or to myelin [79]. As
(EAE) mouse model, and are thought to be due to disruption yet, a final treatment for MS has not been found, and
either of the immune system or of the myelin-producing no therapy has been developed to date for its progressive
cells. As a matter of fact, the hallmarks of MS are inflam- forms, even though RR-MS can count on several disease-
mation and neurodegeneration, where, upon damage of the modifying treatments (DMTs). Besides methylprednisolone,
BBB, massive infiltration of highly proinflammatory and a corticosteroid used immediately after the diagnosis of MS
autoreactive leukocytes occurs (especially T-helper 1 and T- and before any other more specific therapeutic approaches,
helper 17 cells), causing demyelination as well as oligoden- FDA has approved seven other DMTs which are already
drocyte death, axon damage, and even neuronal loss [68]. in commerce: Interferon--1a (IFN--1a), IFN--1b, Glati-
These autoimmune processes are paralleled by continuous ramer acetate, Mitoxantrone, Teriflunomide, Fingolimod,
activation of resident macrophages/microglia that potentiate and Natalizumab [16]. Such drugs are all immunomodulatory
the inflammatory response by producing proinflammatory and aim at halting the pathological immune responses by
cytokines and chemokines, as well as reactive oxidants [69]. directly inhibiting cell activation and the release of proinflam-
The autoimmune and inflammatory hypothesis dominated matory mediators or by limiting cell transmigration into the
the MS research field for almost 50 years until the early 2000s; CNS; yet they still do not represent a definitive solution to the
however, whether inflammatory demyelination is primary problem, especially for the 1520% of MS patients affected
or secondary in the disease process is yet unclear. Indeed, by the progressive forms of MS. Given the involvement
over the last decade, the concept of a neurodegenerative of ROS and RNS in MS pathogenesis, it is possible that
and microglia-centered view has been gaining increasing antioxidant compounds could play a pivotal role in the
attention. In this view, MS might be primarily a neurodegen- prevention of the free radical-mediated tissue damage as well
erative disease with secondary inflammatory demyelination, as inhibiting the early proinflammatory events, such as T-cell
whose trigger starts in the brain and the progress of which is activation and CNS infiltration, which would ultimately lead
modified and amplified by inflammation [70, 71]. Accumu- to brain inflammation and neuronal death [16]. Treatment
lated evidence indicates that oxidative stress plays a major with antioxidant could theoretically prevent the spreading of
role in the pathogenesis of MS. ROS and RNS are mainly tissue damage promoting cellular survival, thus ameliorating
generated in excess by activated microglia and have been the disease outcome (Table 1). In this context, the antioxidant
implicated as mediators of demyelination and axonal damage compound tirilazad mesylate, a member of lazaroid family
typical of MS [72]. In addition, free radicals can activate known for its peroxyl-radical-scavenger properties and for
certain transcription factors, like NF-B, which upregulate its ability to reduce iron-catalysed lipid peroxidation, has
the expression of many genes involved in human MS and been proven useful in preventing the onset of acute EAE
EAE, including TNF-a, iNOS, ICAM-1, and VCAM-1 [73]. (the animal model of MS), as well as reducing its severity.
Additionally, redox reactions are involved in the activity of Another study showed that the administration of NAC,
matrix metalloproteinases (MMPs), which are important to a molecule particularly efficient in boosting intracellular
T-cell trafficking into the CNS [74]. Several studies have levels of GSH, is able to hinder the induction of acute
found evidence of lipid peroxidation in the CSF and plasma EAE [80]. On the other hand, Euk-8, a synthetic salen-
of MS patients, with higher concentration of isoprostanes manganese complex, could emerge as a key compound in the
and MDA. Further, a weakened cellular antioxidant defense, development of a brand new class of molecules possessing
especially due to impairment of SOD, GR, and GPx, as well scavenging properties along with superoxide dismutase and
as elevated levels of GSSG and reduced vitamin E : lipid catalase abilities. As a matter of fact, repeated injections of
ratio, was found in red blood cells of these subjects [75]. Euk-8 upon encephalomyelitis induction in mice were able to
Moreover, direct examination of MS plaques revealed an delay the onset of EAE symptomatic phase as well as reduce
increase in free radical activity and decreased levels of the severity of the clinical phenotype, giving hope to the
relevant antioxidants like GSH, a-tocopherol, and uric acid hypothesis of its possible application in human MS [81]. -
[18]. Furthermore, activated mononuclear cells of MS patients Lipoic acid, an antioxidant molecule capable of crossing the
produce high amounts of ROS and NO , and oxidative BBB, has also been recently reported to suppress inflamma-
damage to DNA (mitochondrial DNA included) develops in tion, demyelination, and axonal damage in EAE mice. Its
association with inflammation in chronic active plaques [76]. effects are mediated by the reduction of T-cell traffic in the
Another study documented that oxidative damage of CNS spinal cord, possibly through the inhibition of MMPs activity.
was provoked by the release of iron from injured cells and Uric acid too has been proven to affect EAE clinical outcome,
by low levels of enzymatic and nonenzymatic antioxidants especially ameliorating neurologic deficits in treated mice,
(particularly ubiquinone and vitamin E) in plasma and in a mechanism involving its ability to inhibit iNOS along
lymphocytes of MS patients [77]. The CNS damage induced with NO and ONOO scavenging properties [82]. However,
by low levels of antioxidants or high levels of ROS might other authors reported that inhibition of NO production is
be caused by the fact that lower levels of antioxidants may instead deleterious in EAE, leaving a wide-open debate on the
promote increased activity of lipoxygenase (that catalyzes one true efficacy of this compound. Some other encouraging data
branch of the arachidonate cascade) [78], thereby increasing obtained from EAE mice led the scientific community to the-
the immunoinflammatory processes within the brain, and orize that the dietary income of antioxidant, such as vitamin
E or selenium, could somehow hinder the progression of MS. Nrf-2, which in turn triggers several cellular antioxidant
Of note, despite the relative abundance of reports describing pathways, resulting in anti-inflammatory and neuroprotec-
the ameliorative effects of restrictive dietary regimens on tive responses [35]. The detailed action is based on the Nrf-
the clinical outcome of affected patients, we still lack a true 2-induced expression of proteins that regulate intracellular
and solid body of evidence supporting the actual action of antioxidant systems such as NQO1, heme-oxygenase-1 (HO-
antioxidant in slowing the progression of MS [83]. Natural 1), glutathione S-transferase Mu-1 (GSTM1), Prx1, and Trx,
occurring molecules currently being investigated in phase I along with a vast number of heat shock proteins (HSPs),
and II trials include polyphenols (especially Gingko biloba thus promoting immune cell survival even in the presence
extracts), essential fatty acids/-lipoic acid, and vitamin of high ROS and RNS concentrations [36]. Thus, Nrf-2
E/selenium [84]. The results of these promising studies will orchestrates a complex machinery that protects neurons and
lay the foundation for phase III trials, which will be pivotal glial cells against the oxidative stress-induced cell damage.
in establishing the long-term efficacy of antioxidant therapies Moreover, a great deal of evidence suggests that DMF is
on MS. An ongoing phase I-II trial is recruiting patients in also able to modulate immune responses either through the
order to assay the effects of idebenone, a synthetic compound Nrf-2-dependent inhibition of the redox signals governed
chemically related to coenzyme Q10. Currently gathered data by NF-B and/or skewing Th1/Th2 balance towards Th2 by
suggest that idebenone could be able to stop demyelination directly inducing T-cell apoptosis, without however resulting
and neuronal death. Another study is also investigating the in immunosuppression. DMF has been also shown to protect
efficacy of -3 PUFAs included in fish oil such as eicosapen- neural stem/progenitor cells and neurons from oxidative
taenoic acid (EPA) and docosahexaenoic acid (DHA) as anti- damage through Nrf2-ERK1/2 MAPK pathway [85, 86]. As
inflammatory, antioxidant, and neuroprotective agents [24]. a consequence, BG-12, a specific oral preparation of DMF,
MS patients treated with fish oil (4 g/die) showed a significant has already been showed in two phase III trials as being
reduction of the levels of proinflammatory cytokines and able to reduce relapse episodes as well as to delay disease
NO catabolites, but no variation in the serum levels of progression in patients affected by RR-MS [8789]. In these
lipoperoxides or the number of relapses per year [25]. The trials, BG-12 was well tolerated, the most common side effects
results coming from such studies are laying the foundations being characterized by redness, gastrointestinal symptoms,
for a phase III clinical trial that should give information and headaches. Another phase III clinic trial is currently
about the long-term efficacy of this strategy on the number investigating BG-12 long-term safety profile with the main
of relapses. The use of antioxidants, even in combination aim of using it in the future as a first-line DMT.
with conventional immunomodulatory therapies, could have
synergistic effects on the disease, resulting in a more powerful 6. Hereditary Spastic Paraplegia
therapy. Indeed, in order to evaluate the real benefit of antiox-
idant therapies on MS patients, adequately designed clinical Hereditary spastic paraplegia (HSP) includes a large and
studies will be needed in conjunction with observational diverse group of genetic disorders whose main feature is
investigations that will evaluate on sufficiently large cohorts progressive spasticity and weakness in the lower limbs, as a
of patients the long-term effectiveness of this potential treat- result of continuous distal axonopathy caused by defects in
ment. The pivotal role of oxidative stress in MS pathogenesis the mechanisms that transport proteins and substances along
and the idea that a therapeutic strategy could reside in the the axons [90, 91]. At least four autosomal dominant HSPs
control of such a phenomenon is highly supported by the are caused by mutations in genes encoding proteins that are
recent approval of the first redox-modulating drug in the involved in ER morphogenesis and that bear an intramem-
treatment of multiple sclerosis. This compound, dimethyl brane hairpin loop responsible for the curvature of ER mem-
fumarate (DMF), which is a methyl ester of fumaric acid, is branes and for their reciprocal interactions. These include
the only oral administration DMT to be approved by both spastin, the most commonly mutated protein in HSP, atlastin-
FDA and the European Medicines Agency (EMA), with the 1, REEP1, and RTN2 [9193]. To date, 72 different spastic gait
trade name Tecfidera. This compound was initially used in disease loci have been identified, and 55 spastic paraplegia
the treatment of psoriasis, though administered as a different genes (SPGs) have already been cloned, most of which play
preparation; however, it was later proposed as therapeutic a role in intracellular trafficking [94]. The products of these
drug for MS due to the immunopathogenic features these two genes are all implicated in disease onset of many forms of HSP
diseases share. Right after administration, the small intestine and can be grouped into functional modules in which they
esterases readily hydrolyze DMF into mono-methyl fumarate are part of specific molecular pathways or perform similar
(MMF), which is more stable and possesses a 12-hour in vivo functions, including dysfunctional axonal transport, axon
half-life, higher than its precursor. The mechanism of action development, dysregulation of myelination, and abnormal
of DMF is based on its ability to interfere with the redox- cellular signaling in protein morphogenesis. Oxidative stress
regulating cellular systems and the consequent modulation is, in fact, one of these functional modules inasmuch as many
of intracellular thiols, which in turn boosts GSH levels. In of these genes affect mitochondrial function and thus deter-
detail, DMF interacts with Kelch-like erythroid cell-derived mine increase in ROS and RNS production, [95]. Indeed,
protein with cap n collar homology-associated protein 1 several reports suggest that oxidative stress could be strictly
Keap-1 at the level of its critical cysteine residue Cys151 involved in the pathogenesis of many forms of HSP. In this
which is covalently adducted, leading to cleavage of this context, one of the most studied genes is paraplegin (SPG7),
protein and the subsequent translocation and activation of a mitochondrial metalloprotease belonging to the family of
ATPases associated with diverse cellular activities (AAA) [96, large number of reactive species and the multiple routes for
97], whose mutations result in mitochondrial dysfunction of their metabolism that simultaneously occur and that interact
muscle tissue and mitochondrial-dependent impairment of with each other, the overlapping of common signaling redox
axonal transport [98, 99]. HSP fibroblasts of patients affected pathways in health and disease, and the scarce knowledge of
by SPG7 show reduced complex I activity in mitochondria their mechanistic insights in vivo. Furthermore, it must be
and an increased sensitivity to oxidative stress [100]. Such considered that the main problem regarding the treatment of
dysfunction could directly contribute to neurodegeneration neurodegenerative diseases is embodied by the necessity of
via free radical mechanism by direct ROS production and developing compounds that are able to cross the BBB, that is,
by a decreased ATP synthesis leading to energy failure. Of the main obstacle between the CNS milieu and the peripheral
note, HSP cells are more sensitive to DNA damage induced by bloodstream. The BBB is able to reduce the efficacy of antiox-
H2 O2 treatment [101], also because abnormal DNA repair is idant drugs as well as several other compounds, which may
another functional module associated with HSP. Also another exert therapeutic action. Consequently, future development
form of HSP, that is, SPG13, is caused by mutations in the of antioxidant therapeutics will undoubtedly depend on a
HSPD1 gene that encodes heat shock protein 60, which is wider knowledge of the BBB-associated transport mecha-
crucial for the folding of several mitochondrial proteins, nisms. Yet, targeting specific oxidative stress pathways, rather
once again affecting mitochondrial function [102]. Further, than the use of dietary antioxidants, seems to represent a
abnormal lipid metabolism is another key functional module novel avenue of research in the management of neurode-
and is also associated with oxidative stress. For instance, generative diseases, especially after the recent approval of
mutations in DDHD1 and CYP2U1 genes, which code for dimethyl fumarate by both the FDA and EMA which acts by
two enzymes involved in fatty-acid metabolism, cause alter- enhancing the antioxidant responses, ultimately promoting
ation of mitochondrial architecture and bioenergetics with cytoprotection of neurons and glial cells.
increased oxidative stress, accounting for lipid metabolism
as a critical HSP pathway with a deleterious impact on Conflict of Interests
mitochondrial bioenergetic function [103]. Overall, these
lines of evidence suggest that oxidative stress is likely a The authors declare that there is no conflict of interests
crucial biomarker and a novel pathogenic mechanism for regarding the publication of this paper.
these neurodegenerative disorders. No specific treatments
are yet available to prevent, slow, or reverse HSP. The avail- Acknowledgments
able therapies mainly deal with management of symptoms
and physical and emotional promotion and include drugs This work was supported by the Italian Foundation of
for muscle tone and spasms as well as antidepressants for Multiple Sclerosis (Grant FISM 2013/R/8 to VC), the Italian
patients experiencing clinical depression. Physical therapy Ministry of Education, University, and Research (Grant PRIN
is also used to restore and maintain the ability to move. 2011 to MM), the Italian Ministry of Health (Grant GR09.109
The probable implication of mitochondrial dysfunction and to AO), and the University of Rome Tor Vergata (Grant
oxidative stress in the pathophysiology of HSP set the basis E82I15000190005 to AO).
for the development of novel therapeutic strategies focused
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http://dx.doi.org/10.1155/2016/3187560
Research Article
Evidence for Detrimental Cross Interactions between
Reactive Oxygen and Nitrogen Species in Lebers Hereditary
Optic Neuropathy Cells
Micol Falabella,1 Elena Forte,1 Maria Chiara Magnifico,1 Paolo Santini,1

Marzia Arese,1 Alessandro Giuffr,2 Kristina RadiT,3 Luciana Chessa,4 Giulia Coarelli,5
Maria Chiara Buscarinu,5 Rosella Mechelli,5 Marco Salvetti,5 and Paolo Sarti1
1
Department of Biochemical Sciences and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy
2
CNR Institute of Molecular Biology and Pathology, 00185 Rome, Italy
3
Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
4
Department of Clinical and Molecular Medicine, Sapienza University of Rome, 00185 Rome, Italy
5
Centre for Experimental Neurological Therapies, S. Andrea Hospital-Site, Department of Neuroscience,
Mental Health and Sensory Organs (NESMOS), Sapienza University of Rome, 00185 Rome, Italy
Correspondence should be addressed to Paolo Sarti; paolo.sarti@uniroma1.it
Received 10 August 2015; Revised 19 October 2015; Accepted 25 October 2015
Academic Editor: Liudmila Korkina
Copyright 2016 Micol Falabella et al. This is an open access article distributed under the Creative Commons Attribution License,
Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species
bioavailability can contribute to cell dysfunction in Lebers hereditary optic neuropathy (LHON) patients. We report that peripheral
blood mononuclear cells (PBMCs), derived from a female LHON patient with bilateral reduced vision and carrying the pathogenic
mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed
by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays
with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure,
leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important
role in driving LHON pathology when excess NO remains available over time in the cell environment.
1. Introduction clinically relevant phenotype in most of the patients is RGCs

loss, seldom accompanied by other complicating neurolog-
Lebers hereditary optic neuropathy (LHON) is a mito- ical disorders, such as dystonia, multiple sclerosis- (MS-)
chondrial disorder leading to severe visual impairment, due like illness parkinsonism, cerebellar ataxia, and myoclonus
to retinal ganglion cells (RGCs) death and atrophy with [5], pointing to a diffused mitochondrial energetic failure.
demyelination of the optic nerve [1]. The primary cause of Interestingly, a higher risk of developing MS in women
the disease is a mitochondrial genome (mtDNA) mutation with clinically established LHON has been reported [6
leading to a single amino acid substitution in one of the mito- 8]. Rarely other phenotypes have been described, such as
chondrially encoded subunits of NADH:ubiquinone oxidore- chronic renal failure [9, 10], involving as common feature
ductase, complex I of the electron transport chain (ETC). tissues/cells that are exquisitely energy dependent and require
The most common mutations are at positions 11778/ND4, adequate supply of reducing substrates and O2 to sustain
3460/ND1, and 14484/ND6 [2, 3]. mitochondrial adenosine-5 -triphosphate (ATP) production.
LHON pathology generally occurs in the second or third All LHON mutations induce an impairment of mito-
decade of life and affects predominantly males [4]. The only chondrial function. A decline in complex I-sustained cell
respiration and ATP production has been reported in assays NO levels, as those produced by iNOS, in combination with
performed on isolated mitochondria derived from muscle, ROS overproduction can result into mitochondrial defects,
Epstein-Barr Virus- (EBV-) transformed leukocytes, periph- eventually triggering severe cell dysfunctions.
eral blood mononuclear cells (PBMCs), and cybrids [11 Here, we tested this hypothesis on PBMCs and lym-
15]. Based on these specific defects, experiments carried out phoblasts derived from a female LHON patient with bilateral
on patients tissues and cell models of the disease showed reduced vision and immunological disorders. We found
an overproduction of reactive oxygen species (ROS) and that the cells, although carrying the pathogenic mutation
an increased propensity to apoptotic cell death [16]. Nev- 11778/ND4, are still endowed with a suitable bioenergetic
ertheless, LHON cells seem to be able to cope with the apparatus, producing ATP, thus well compensating complex
ETC dysfunction, maintaining apparently a normal growth I mutation. However, lymphoblasts proved to be more sus-
and total cellular ATP level, yet being more vulnerable to ceptible to NO toxicity, suggesting that the mitochondrial
metabolic/oxidative stress or other stressful conditions [2, 17]. genetic defect, via the enhancement of the basal ROS levels,
Consistently, a recently developed mouse model revealed, potentiates the formation of secondary reactive nitrogen
together with some of the key histopathological features species (RNS), particularly ONOO , leading to reduced cell
typically observed in LHON patients, the decrease of complex viability.
I activity, respiratory defects, and the increase in ROS levels,
but no reduction in ATP synthesis, pointing to oxidative 2. Materials and Methods
stress as the major driver of the pathology [18]. On these
bases, most of therapeutic approaches to LHON, and gen- 2.1. Chemicals. RPMI-1640 medium, fetal bovine serum (FBS),
erally to mitochondrial disorders, currently rely on the and nonessential amino acid solution were from Sigma-Aldrich
use of mitochondrial substrates, together with redox active and Invitrogen Life Technologies (GIBCO). Thiazolyl blue
effectors and free radical scavengers. Idebenone, curcumin, tetrazolium bromide (MTT) was from Sigma-Aldrich and
and vitamins C and E as well as other antioxidant compounds (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-
have been used separately or combined in cocktails cus- 1-ium-1,2-diolate (DETA-NONOate) was purchased from
tomized for the individual patients [19, 20], all treatments Cayman Chemicals.
unfortunately with limited success.
Under severe pathological conditions, such as inflamma- 2.2. Case Report. The proband is 34-year-old woman with
tion or sepsis, a disruption of the homeostatic control of oxy- a previous diagnosis of autoimmune thyroiditis. When she
gen supply and utilization has been observed, accompanied was 28 years old, she suffered a sudden and painful loss
by ROS formation, together with an imbalance of nitric oxide of vision in OS and after two weeks in OD, accompanied
(NO) production and breakdown. Nitric oxide produced by xerophthalmia. Visual field showed retinal sensibility
either enzymatically by nitric oxide synthases (NOSs) [21] reduction, especially at superior areas, whereas fluoroan-
or directly via the reduction of bulk nitrite at low pH [22] giography was normal. She was treated with high doses of
is a fundamental second messenger involved in a number corticosteroids and intravenous immunoglobulins without
of pathophysiological processes [23], inducing detrimental benefits. Brain MRI scan was unremarkable. After 4 months,
or cytoprotective effects depending on its concentration and plasmapheresis was performed with little improvement, so
localization [24]. Importantly, NO regulates mitochondrial it was decided to start cyclophosphamide treatment. This
respiration by reversibly binding to cytochrome oxidase treatment was carried on for 6 months without any benefit.
and limiting O2 consumption, while extending O2 gradients In the meantime, after a femur fracture due to an accidental
in tissues [25, 26]. Such inhibition is more effective in fall, a severe osteoporosis was diagnosed. After one year from
actively respiring cells and at low oxygen concentration [27 the visual loss, a workout for autoimmune disorders gave
29]. Under conditions associated with increased ROS levels, positive ANA (speckled pattern, 1 : 160) and the presence
NO participates in reactions with other reactive species to of anti-Ro/SSA. Cytochemical, bacteriological, viral, and
generate secondary products that can impair mitochondrial immunoelectrophoretic analyses on cerebrospinal fluid were
function. Particularly, the reaction of NO and superoxide normal.
anion (O2 ), leading to the formation of toxic peroxynitrite Brainstem auditory evoked responses, along with motor
(ONOO ), is very rapid (diffusion limited) and known to and somatosensory evoked potentials, were normal, while
induce macromolecular damage, including nitration and visual evoked potentials could not be performed due to
inactivation of mitochondrial proteins [30]. bilateral visual loss. A new brain and orbital and spinal
Although many biochemical aspects of LHON have been cord MRI scan showed bilateral callosal, periventricular, and
elucidated, the role of NO in the LHON disease has not been paratrigonal white matter symmetric hyperintensities on T2-
investigated yet. Interestingly, an increased immunoreactivity weighted images without contrast enhancement on T1. No
for inducible nitric oxide synthase (iNOS) has been detected alterations were found at the level of the spinal cord and
in macrophages and in the microglia of demyelinated lesions optic nerves. On admission to our neurological division,
in the brain white matter of a LHON female patient, suggest- bilateral visual acuity was 1/10. We excluded neoplastic or
ing an early immunological mechanism in addition to the paraneoplastic processes by full-body CT scan and screening
primary degeneration of the optic nerve [31]. In this patient, for antibodies associated with paraneoplastic syndromes.
administration of corticosteroids improved visual and neu- The infectious aetiology was also discarded after negative
rological function. This observation suggests that increased hepatitis B and hepatitis C virus, HIV, EBV, cytomegalovirus
serology, and tuberculin skin test. Neuromyelitis optica, (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL
carential, metabolic, and endocrinological disorders were streptomycin, and 1X nonessential amino acids. Cells were
also excluded. The standard genetic screening for LHON cultured in 25 cm2 or 75 cm2 flasks or in multiwell plates and
disease showed the presence of 11778/ND4 mutation. She incubated at 37 C under standard conditions [5% CO2 ; 95%
began ubidecarenone therapy (600 mg/day) without any sub- relative humidity]. Viable-cell counting was carried out using
jective or measurable improvement of visual acuity. After the Trypan blue dye exclusion test (Sigma-Aldrich).
two months she developed Escherichia coli pyelonephritis and
chronic tubulointerstitial nephritis was diagnosed. After one 2.5. Cell Respiration. Oxygen consumption in intact PBMCs
year and a half from the visual loss, the patient presented a derived from the LHON patient and controls was mea-
bilateral visual acuity worsening with a severe headache and sured using a high resolution respirometer (2k-Oxygraph,
hypoesthesia at four limbs. A brain MRI showed a massive OROBOROS Instruments). Data acquisition and analysis
extension of bilateral paratrigonal white matter lesions with were carried out using the software Datalab (OROBOROS
an involvement of bilateral occipital subcortical white matter Instruments). Control and LHON PBMCs were collected,
and increased signal intensity on T2-weighted images of washed with sterile phosphate buffered saline (PBS), counted,
optical radiations and retrochiasmatic optic tracts. After one and suspended in DMEM containing 1 g/L glucose. Cells,
month, brain MRI with contrast and diffusion weighted
at a final density ranging from 7 to 9 106 cells/mL, were
imaging was repeated showing an additional enlargement of
incubated in the 2k-Oxygraph chambers at 37 C for 30 min
bilateral occipital lesions with positivity in DWI sequence and
to allow temperature and pH equilibration. Respiration was
a circled enhancement. Therefore she was admitted again to
evaluated under basal metabolic conditions, thus sustained
our division and a hemogasanalysis proved a severe metabolic
by endogenous substrates. After recording an oxygen con-
acidosis (pH 7.14, pCO2 = 13 mmHg, pO2 = 145 mmHg,
sumption rate (OCR) baseline, 4 M antimycin A (AA) was
sO2 = 98.5%, HCO3 = 4.40 mmol/L, BE = 22.40 mmol/L,
added to the chamber in order to inhibit cytochrome
and BEecf = 24.60). Blood lactate measurements showed
reductase (complex III) and stop mitochondrial respiration.
an abnormal increase during exercise (at rest 13.69 mg/dL;
Data were recorded at sampling intervals of 2 s.
after 5-minute exercise 48.13 mg/dL; after 10-minute exercise
65.53 mg/dL; after 15-minute exercise 85.57 mg/dL; 15 minutes
after the end of exercise 59.28 mg/dL; and normal value 4.5 2.6. Determination of ATP Levels. Cellular concentration of
19.8). Blood analysis showed a renal injury associated with ATP was measured by chemiluminescence, under stationary
nephrocalcinosis diagnosed by ultrasonography and CT scan. conditions. Cells were incubated overnight in antibiotic/FBS-
During the hospitalization the medical care focused on the free DMEM medium. Steady-state ATP levels were mea-
improvement of electrolyte balance and renal injury until sured in LHON and control PBMCs (3 105 cells/mL) 4 h
values normalization. Simultaneously, by brain MRI scan, after resuspending the cells in PBS containing L-glutamine
we appreciated a clear reduction of white matter lesions and (2 mM), in the presence of glucose (2 g/L) or its absence to
contrast enhancement absence. stimulate OXPHOS; when necessary, oligomycin (2.5 g/mL)
was added over the last 1.5 h of incubation. Synthesized ATP
2.3. Peripheral Blood Mononuclear Cells (PBMCs) Isolation. was measured using the ATPlite 1step kit (PerkinElmer) in
Blood samples were obtained from the case report and from a luminometer (VICTOR Multilabel Counter, PerkinElmer,
three matched (age, sex, and geographical origin) healthy USA) equipped with 96-well plates.
controls. The local Institutional Review Board approved the
study and all participating subjects gave written informed 2.7. Reactive Oxygen Species (ROS) Quantification by Flow
consent. Cytometry. To measure the intracellular ROS concentra-
PBMCs were obtained by density centrifugation over tion, the fluorescent probe 2 ,7 -dichlorofluorescein diac-
Ficoll-Hypaque according to standard procedures. Genomic etate (DCFDA, Sigma-Aldrich) was used. PBMCs from the
DNA was extracted from PBMCs using a commercial kit LHON patient and controls were incubated for 30 min at
(QIAamp DNA Mini Kit, Qiagen). Purified PBMCs (5 104 37 C in the dark in RPMI-1640 medium containing 10 M
cells/well) were seeded in 96-flat well plates and cultured DCFDA. Afterwards, cells were washed twice with Hanks
in 200 L/well RPMI-1640 medium, supplemented with 20% Buffered Salt Solution (HBSS) supplemented with calcium
FBS (HyClone), 2 mM L-glutamine, 100 U/mL penicillin, and and magnesium, as suggested by the manufacturer, collected,
100 g/mL streptomycin. and analysed immediately by flow cytometry (BD Accuri
C6); ROS levels were estimated from the mean fluorescence
2.4. Preparation and Cultures of Lymphoblast Cell Lines. Lym- intensity. The green fluorescence was measured using the FL-
phoblast cell lines were established from peripheral blood 1 setting (log mode) after having gated out cell debris. In each
PBMCs of the patient and the three healthy controls by EBV experiment 10.000 events were recorded.
infection. The mutational analysis of the patient mtDNA was
performed by PCR, restriction analysis, and electrophoresis 2.8. 3-Nitrotyrosine Level Detection. The content in 3-nitro-
and confirmed by sequencing (Sanger method). Both PBMCs tyrosine (3-NT) modified proteins was used as marker of
and lymphoblast cell lines derived from the LHON patient protein damage by ONOO . The intracellular 3-NT levels
and controls were grown in RPMI-1640 medium supple- were assessed colorimetrically using a competitive ELISA kit
mented with 20% (v/v) heat-inactivated fetal bovine serum (Abcam). For each independent experiment a standard curve
was generated with the provided 3-NT standard and the 3-NT PBMCs, suggesting that both cell types possess an effi-
content quantified. cient glucose-dependent glycolytic compensation (Warburg
effect). Without glucose (Figure 1(c)), both LHON and
2.9. Nitrate/Nitrite (NOx ) Determination. NOx concentration control cells showed 20% decrease in the ATP content,
in cell supernatants was determined fluorimetrically using whose concentration dropped dramatically in the presence
Fluorimetric Assay Kit (Cayman Chemical), 48 h after cells of oligomycin. Under the latter conditions, the glycolytic
seeding. contribution is minimal and the difference between the ATP
measured in the presence and absence of oligomycin (ATP)
is indicative of the ATP generated by OXPHOS (ATPOXPHOS ).
2.10. Cell Viability Assay. After 48 h incubation in presence or
The ATPOXPHOS /ATPTOTAL ratio (Figure 1(d)) is very similar
absence of the NO donor DETA-NONOate, the viability of
in LHON and control cells, indicating that the mutation does
lymphoblast cells was assessed using the MTT [-3(4,5-dim-
not affect OXPHOS efficiency.
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduc-
Taken together, these results suggest that, over and above
tion assay as described in [32]. Briefly, cells (2 105 /mL) were
a bioenergetic deficit imputable to complex I mutation, other
seeded in a 96-well plate and incubated for 48 h at 37 C in
molecular mechanisms contribute to the clinical onset of
the presence of increasing DETA-NONOate concentrations
LHON and its progress that we hypothesize to be also
(0.010.5 mM) in a final volume of 100 L/well. Having a
related to increased cell levels of reactive oxygen and nitrogen
half-life of 20 h at 37 C, the NO donor was readded after
species.
24 h. At the end of the 48 h incubation, 10 L of MTT
solution (5 mg/mL) was added to each well, followed by
3.2. Increased Level of ROS, 3-Nitrotyrosine, and Nitrite in
4 h incubation at 37 C. Afterwards, in order to dissolve
PBMCs. In order to investigate whether the PBMCs derived
the dark-coloured formazan crystals produced by reduction
from the LHON patient displayed increased oxidative/
of the MTT tetrazolium salt, cells were incubated at 37 C
nitrosative stress levels, we measured the amount of ROS
overnight with 100 L of 10% sodium dodecyl sulphate (SDS)
and 3-NT under basal conditions. The latter is an important
in 0.01 M HCl. The optical density of reduced MTT was
marker of RNS, including ONOO . We found that LHON
measured at 570 nm with a reference wavelength at 690 nm
PBMCs display a significant 1.4-fold increase in ROS produc-
using Appliskan Microplate Reader (Thermo Scientific). The
tion compared to controls (Figure 2(a)). Consistently, LHON
experiments were carried out in triplicate.
PBMCs displayed a higher concentration of 3-NT, suggesting
a role of NO chemistry in cellular stress (Figure 2(b)).
2.11. Statistical Analysis. Data are reported as mean SEM of To assess the basal NO level in LHON, we measured the
at least three independent experiments and significance () concentration of nitrite/nitrate, the oxidation products of
was determined using Students -test. values 0.05 were NO. Interestingly we found that the nitrite/nitrate levels
considered significant. (Figure 2(c)) tend to increase compared to controls, although
the reported variation did not reach statistical significance.
3. Results Owing to a limited availability of biological samples (cells
from patient) and in order to obtain larger amounts of start-
3.1. Mitochondrial Function in LHON Patient PBMCs. RGCs, ing material, EBV-transformed lymphoblasts were used for
brain, and kidneys, the most affected sites in the LHON further cell biochemical analysis. The bioenergetic behaviour
patient, are all high-energy demanding, so they rely more of primary and transformed cells was very similar, with
on OXPHOS ATP. Importantly, PBMCs have a metabolism the two cell types displaying superimposable mitochondrial
mainly sustained by OXPHOS [33, 34] and therefore repre- parameters (data not shown).
sent a good model to be investigated in the present study. Fig-
ure 1(a) shows a typical oxygen consumption trace acquired 3.3. DETA-NONOate Decreases LHON Lymphoblasts Viabil-
with intact PBMCs. The oxygen consumption was sustained ity. A chronic increase in NO levels was artificially mim-
by endogenous substrates and relied almost completely on icked using the NO donor DETA-NONOate (DETA-NO).
mitochondria, as it was almost completely inhibited by Cell viability was examined in both LHON and control
antimycin A, a specific inhibitor of cytochrome reductase. lymphoblasts after 48 h incubation with different amounts
After normalization to the protein content, we found the O2 of the NO releaser. As a regulator of cell proliferation, NO
consumption rate (50 pmol O2 s1 mg1 ) to be very similar can either enhance or inhibit cell growth depending on
in both LHON and control cells. its concentration. Figure 3 shows that at 10 M DETA-NO
In agreement with these data, no significant differences no cytotoxicity was associated with NO exposure. Higher
in the steady-state concentration of ATP were observed in concentrations of DETA-NO affected cell viability propor-
control and patient PBMCs. ATP levels were evaluated either tionally to the amount added. However, the decline in cell
in the absence or in the presence of glucose to sustain viability was significantly larger in LHON than in control
glycolysis and using oligomycin to inhibit OXPHOS activity. cells: at 0.1 and 0.5 mM DETA-NO, the residual viability
When glucose was present (Figure 1(b)), both LHON and of LHON cells was 35% and 15%, respectively, while in
control cells displayed a similar ATP content (ATPTOTAL control cells it was, respectively, 75% and 35%. Trypan blue
6 g ATP/mg total protein), and oligomycin inhibition of exclusion assays, carried out under comparable experimental
OXPHOS was found poorly effective in control and LHON conditions, indicated a similar cellular behaviour (data not
300 500
250
O2 slope (pmol s1 mg1 )

400
O2 concentration (M)
LHON Control
200
300
150
AA 200
100 AA
50 100
0 0
0 1000 2000 3000 4000
Time (s)
(a)
8 8
1
6 6
ATP OXPHOS /ATP TOTAL

(g ATP mg1 )
(g ATP mg1 )
0.8
4 4
0.6
ATP ATP
2 2 0.4
0.2
0 0
Glucose + + + + Glucose
Oligomycin + + Oligomycin + + 0
Control Control Control

LHON LHON LHON
(b) (c) (d)
Figure 1: Bioenergetic properties of 11778/ND4 mutated PBMCs. (a) Representative cell O2 consumption measurements. Black line: O2
concentration trace; red line: O2 consumption rate. After recording basal respiration, 4 M antimycin A (AA) was added. (b) Cellular ATP
levels measured in the presence of glucose with and without oligomycin. (c) Cellular ATP levels measured in the absence of glucose with and
without oligomycin. (d) Fractional ATP expressed as the ratio between ATPOXPHOS and ATPTOTAL . ATPOXPHOS was obtained from ATP
(c) and ATPTOTAL was measured in the presence of glucose without oligomycin (b). Data (mean SEM) were collected in three triplicate
experiments on cells derived from the LHON patient and the three healthy controls.
300.000 150 300 NS

250.000
1
3-Nitrotyrosine (nM mg )
200.000
NOx (M mg1 )
100 200
Mean (a.u.)
150.000
100.000 50 100
50.000
0 0 0
Control Control Control

LHON LHON LHON
(a) (b) (c)
Figure 2: Oxidative and nitrosative stress is increased in 11778/ND4 mutated PBMCs. (a) Intracellular ROS levels were measured using the
DCFDA dye. (b) The 3-NT content was determined by competitive ELISA and normalized to total protein. (c) Concentration of NOx (nitrite
and nitrate) in the cell supernatant as normalized to total protein. Data (mean SEM) collected in three duplicate experiments on cells derived
from the LHON patient and the three healthy controls. Values are considered significant when < 0.05: 0.05; Ns: not significant.
120 mitochondrial overexpression of MnSOD induces a decrease

in superoxide levels and enhances cell survival [39]. While
Cell viability (% of control)
100 chronic oxidative stress has been shown to play an important

80 role in the onset of the disease, the role of NO and related
reactive species remained unexplored. Together with CO and
60
H2 S, NO forms the gasotransmitter triad acting as a redox-
40 signalling regulator of several physiological functions, the
mitochondrial one included [40]. NO, particularly at low
20
concentrations, protects against cell death [4144], whereas
0 in the presence of superoxide it becomes toxic by forming
0 0.01 0.05 0.1 0.5 ONOO [30]. It should be kept in mind, however, that, even
DETA-NO (mM) in the absence of ONOO , depending on the electron flux
Control level through the respiratory chain, that is, on the concen-
LHON tration of mitochondrial ferrocytochrome , and particularly
under low O2 tension (hypoxia), inhibition of mitochondrial
Figure 3: NO impairs the viability of 11778/ND4 mutated lym- complex IV by NO can be severe and persistent [29, 45, 46].
phoblasts. The cells derived from the LHON patient and the three
According to our results, LHON cells are apparently
healthy controls were treated with various concentrations of DETA-
NONOate for 48 h and their viability was assessed using the MTT
more prone to such NO-dependent detrimental chemistry.
assay. Data are shown as the percentage of cell viability measured We present here the case of a female patient with a point
in the absence of the NO releaser. Data acquired in three triplicate mutation at nucleotide position G11778A, who suffered from
experiments are expressed as mean SEM. Values are considered both eyes visual loss and subsequently displayed markers for
significant when < 0.05: 0.05; 0.01. autoimmune disorders, as well as white matter alterations
outside the visual system.
We show that PBMCs and lymphoblasts derived from
shown), showing that LHON cells are more susceptible to our patient are more susceptible not only to oxidative but
nitrosative stress than control cells. also to nitrosative stress. In these cells we found that,
under metabolic basal conditions and in the absence of
4. Discussion exogenous NO, the 3-NT levels are enhanced. This finding is
in agreement with prior studies carried out on optic nerve
LHON is characterized by a low penetrance, as most indi- and retinal histological specimens obtained from LHON
viduals carrying mtDNA mutations remain asymptomatic, patients and on synaptosomes from a mouse model of the
with a limited subset of them expressing the disease. Age disease that have evidenced increased levels of 3-NT [18, 47].
and gender are the most important risk factors, with approx- The higher basal 3-NT concentration suggests that LHON
imately 3050% of males and 1020% of female LHON cells produce larger amounts of nitrosating agents, such
mutation carriers becoming symptomatic at a median age of as ONOO . Consistently, we found that the concentration
19 years (range 556 years) [35]. Indeed, genetic, epigenetic, of nitrite/nitrate, the oxidation products of NO, tends to
and environmental factors all contribute to the onset and increase in patient cells, compared to controls. Increase
evolution of the disease [36]. Complex I mutation, the in nitrosative stress might be particularly likely when NO
primary etiologic cause of LHON, despite being a necessary overproduction takes place.
determinant of the pathology, is not sufficient to induce The current study reveals that chronic exposure to NO
the clinical expression of the disease so that a convincing in proband lymphoblasts carrying the 11778/ND4 mutation
explanation for the pathogenesis of this disorder is still under determines a significant decrease in cell viability compared
debate. The impairment of bioenergetics parameters, such to controls, suggesting that in this pathological state the NO
as cell respiration and ATP production, has been found chemistry is more active. Interestingly, our patient has shown
absolutely modest both in patients tissues and in cell models an abnormal increase in lactate serum levels after exercise,
of the disease. On the contrary, a marked overproduction suggesting a limited mitochondrial reserve capacity. In terms
of ROS has been reported, together with a low capability of energy metabolism, under basal conditions the patient
of the cellular antioxidant machinery to maintain the redox PBMCs are able to fully compensate complex I mutation
homeostasis. allowing an efficient mitochondrial function. We observed
LHON cybrid models have shown lower levels of glu- that the ATP levels in LHON cells were not significantly
tathione peroxidase, glutathione reductase, and Mn-superox- different from those ones of control cells, even when the
ide dismutase (MnSOD) or total glutathione, compared to glycolytic system was restricted by glucose deprivation.
controls [37, 38], particularly after cell treatment to enhance Both cell types were equally able to efficiently compensate
their OXPHOS dependency. These observations strongly OXPHOS defects with glycolysis, as shown by abolishing the
support the idea that the genetic alteration of complex I, contribution of ATPOXPHOS with oligomycin.
over and above the simple action on primary mitochondrial Mitochondrial ATP generation is crucial for cell func-
parameters, induces an alteration of the cell redox-signalling tion; therefore mitochondria have evolved several different
and homeostasis. Interestingly, in a cybrid model carry- strategies to maintain it to face dysfunctions. Switching
ing the G11778A/ND4 mutation, it has been observed that to glycolysis is fundamental for cell survival during acute
stress, although this pathway represents a much less efficient References

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http://dx.doi.org/10.1155/2016/7239639
Review Article
Exercise Modulates Oxidative Stress and Inflammation in
Aging and Cardiovascular Diseases
Nada Sallam1 and Ismail Laher2

1
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
2
Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia,
Vancouver, BC, Canada V6T 1Z3
Correspondence should be addressed to Ismail Laher; ilaher@mail.ubc.ca
Received 14 August 2015; Accepted 28 September 2015
Copyright 2016 N. Sallam and I. Laher. This is an open access article distributed under the Creative Commons Attribution
cited.
Despite the wealth of epidemiological and experimental studies indicating the protective role of regular physical activity/exercise
training against the sequels of aging and cardiovascular diseases, the molecular transducers of exercise/physical activity benefits are
not fully identified but should be further investigated in more integrative and innovative approaches, as they bear the potential for
transformative discoveries of novel therapeutic targets. As aging and cardiovascular diseases are associated with a chronic state
of oxidative stress and inflammation mediated via complex and interconnected pathways, we will focus in this review on the
antioxidant and anti-inflammatory actions of exercise, mainly exerted on adipose tissue, skeletal muscles, immune system, and
cardiovascular system by modulating anti-inflammatory/proinflammatory cytokines profile, redox-sensitive transcription factors
such as nuclear factor kappa B, activator protein-1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha,
antioxidant and prooxidant enzymes, and repair proteins such as heat shock proteins, proteasome complex, oxoguanine DNA
glycosylase, uracil DNA glycosylase, and telomerase. It is important to note that the effects of exercise vary depending on the type,
intensity, frequency, and duration of exercise as well as on the individuals characteristics; therefore, the development of personalized
exercise programs is essential.
1. Exercise Training and Aging osteoporosis [36], sarcopenia [37], anxiety [38], and cognitive
impairment [3941]. Most importantly, exercise improves the
There is mounting evidence based on epidemiologic and quality of life of elderly people [42, 43].
experimental studies that physical activity and exercise train-
ing combat the sequels of aging. Physical activity is defined 2. Exercise Training and
as any bodily movement coordinated by skeletal muscles,
which increases energy expenditure over resting condition
Cardiovascular Diseases
[1], whereas exercise training is a more regular and structured Age is a major risk factor for cardiovascular diseases (CVDs)
form of physical activity. Higher levels of physical activity and [44, 45]. Numerous studies, confirmed by meta-analyses,
regular exercise are associated with reduced risks of all-cause indicate that exercise training reduces cardiovascular mor-
mortality [220] and also with increased longevity [18, 21 tality [7, 20, 26, 4654] and cardiovascular events [4, 10, 25
23]. In fact, the World Health Organization has identified 28, 30, 31], particularly stroke [5558], coronary heart disease
physical inactivity as the fourth leading risk factor for global [25, 5961], heart failure [60, 62, 63], atherosclerosis [6466],
mortality [24]. Furthermore, physical activity and exercise and preeclampsia [6769]. Accordingly, physical inactivity is
training reduce the risk of age-associated diseases, namely, now regarded as one of the most prevalent cardiovascular
cardiovascular diseases [4, 10, 2531], type 2 diabetes [32], risk factors [70, 71]. Moreover, exercise training is an effective
metabolic syndrome [33], colon cancer [34], obesity [35], therapeutic strategy for patients with peripheral arterial
diseases [7274], coronary heart disease [7581], heart failure factor-alpha (TNF-), interleukin-6 (IL-6), and interleukin-
[8284], atherosclerosis [64], and hypertension [8588]. 1 beta (IL-1) [148150]. Indeed, aging is associated with
The cardiovascular benefits of exercise have been fre- increased levels of TNF-, IL-6, and interleukin-1 receptor
quently attributed to the reduction of many classical cardio- agonist (IL-1ra) and systemic inflammatory biomarkers such
vascular risk factors including blood lipids [20, 28, 50, 89 as C-reactive protein (CRP) as well as higher count of
95], high blood pressure [20, 28, 50, 95], obesity [50, 95 inflammatory cells such as neutrophil and monocytes [151
97], glucose, and type 2 diabetes [98, 99] as well as novel 153]. Hence, aging is associated with oxidative stress and
risk factors such as inflammation [28, 100103] and oxidative inflammation.
stress [95]. However, the mechanisms underlying the protec- Cardiovascular diseases are also associated with high level
tive and therapeutic effects of exercise go beyond reducing of inflammation and oxidative stress [154157].
cardiovascular risk factors [104] to modulating angiogenesis
[105], endothelial progenitor cells [106109], basal heart rate 4. Oxidative Stress and Inflammation
[110], endothelial function [111115], autonomic control [116], Overlapping Signaling Pathways
arterial stiffness [41, 117120], and arterial remodeling [121].
In this review we will focus on the molecular transducer of Oxidative stress and inflammation share common and over-
the antioxidant and anti-inflammatory effects of exercise. lapping signaling pathways. By damaging macromolecules,
ROS can initiate inflammation [158]; ROS are also products
3. Oxidative Stress and Inflammation in Aging of the inflammatory process. During the respiratory burst,
and Cardiovascular Diseases immune cells generate RONS via NADPH oxidase and iNOS
and release proinflammatory cytokines such as TNF-, IL-
Aging is associated with oxidative stress that is mainly 1, and IL-6 [141, 159, 160]. Similarly, injured tissues can
attributed to defective mitochondria, resulting from reduc- also release proinflammatory cytokines that activate specific
tion in cytochrome C oxidase (complex IV) activity [23, 122, ROS-generating enzymes such as lipoxygenase, NADPH
123] and peroxidative damage of mitochondrial membrane oxidase, myeloperoxidase, and xanthine oxidase [142147]
[124]. Hence, a greater number of electrons are generated that and specific reactive nitrogen species generating pathways
can escape from the mitochondria to create a long trail of such as NOS, protein kinase B (Akt), and Sph1P (sphingosine-
reactive oxygen species (ROS) [125, 126], leading to further 1-phosphate) [161163].
mitochondrial dysfunction and ROS generation and creating ROS overproduction activates redox-sensitive transcrip-
a vicious cycle of oxidative damage [127]. Age-associated tion factors including nuclear factor kappa B (NF-B) and
increases in ROS production occur in skeletal muscles [128] activator protein-1 (AP-1) via stress kinases such as extra-
and other organs such as the heart, liver, brain, and kidney cellular signal regulated kinases (ERKs), c-jun N-terminal
[23, 126, 129, 130]. kinases (JNKs), mitogen activated protein kinase p38 (MAPK
Reduced protein synthesis limits antioxidant defense p38), protein kinase C (PKC), phosphatidylinositol-4,5-
mechanisms and repair capacity in aged individuals, which bisphosphate 3-kinase (PI3K)/Akt, and Src family kinases
further contributes to the state of oxidative stress. The free (SFKs). This leads to increased expression of inflammatory
radical theory of aging hypothesizes that oxidative stress target proteins such as matrix metalloproteinase-9 (MMP-
damages macromolecules, including lipids, proteins, and 9), intercellular adhesion molecule-1 (ICAM-1), vascular
nucleic acids, overwhelming cellular antioxidant defense cell adhesion molecule-1 (VCAM-1), iNOS, cyclooxygenase-
and repair mechanisms, leading to progressive deleterious 2 (COX-2), and cytosolic phospholipase A2 (cPLA2) (Lee
changes over time [131, 132]. Indeed, oxidatively damaged and Yang) [164171] and proinflammatory mediators such
proteins [133], nucleic acids [134, 135], and lipids [113, 136 as TNF- gene [172], IL-1, and IL-8 [169]. Many of these
138] are abundant in various organs and tissues such as inflammatory proteins or their products such as NOS, COX,
kidney, liver, heart, arteries, skeletal muscles, and plasma in and PGE2 are prominent sources of RONS [173]; this creates
aged subjects. an autoactivating loop which feeds the vicious cycle of
Aging is also accompanied with a state of chronic inflam- inflammation and oxidative stress.
mation that is mainly attributed to sarcopenia and adiposity. There are also other proteins such as thioredoxin-
Sarcopenia, defined as age-associated progressive loss of interacting protein (TXNIP) linking oxidative stress and
muscle mass and strength [139, 140], increases the incidence inflammation. Under resting conditions, TXNIP is bound to
of muscle injury [18], which increases the infiltration of thioredoxin (TRX) via a disulphide bound, keeping it in an
immune cells into injured muscles. Activated immune cells inactive form. Increased levels of ROS generation cause the
and injured muscles release proinflammatory mediators and dissociation of TXNIP from TRX, leaving it free to scavenge
reactive oxygen and nitrogen species (RONS) via lipoxyge- ROS and allowing TXNIP to stimulate the inflammatory
nase, NADPH oxidase, xanthine oxidase, and inducible nitric cytokine IL-1 [174, 175]. In agreement with this is the obser-
oxide synthase (iNOS) [141147] leading to oxidative stress. vation that antioxidant supplementation blocked the anti-
Sarcopenia can also lead to reduced physical activity and inflammatory effect of exercise by reducing IL-6 production
increased adiposity. Adiposity induces a state of low-grade [176, 177].
but chronic inflammation through the release of a multi- In short, proinflammatory mediators such as TNF-, IL-
tude of proinflammatory cytokines including tumor necrosis 1, and IL-6 generate RONS which activate redox-sensitive
e, Aging
as
id
ER
ox
Ks
H RONS
, JN
PI
DP XO Oxidative stress Exercise Exercise
3K
Exercise
A O,
Ks
N
/A
, P
,
O
M
kt,
LP ,M
AP
OS
SF
K
iN Defective
Ks
Obesity Sarcopenia
p3
,S
mitochondria
8
ph
P1
Immune cells NF-B and AP-1
Inflammation Inflammation Oxidative stress
Exercise Exercise
COX-2, PGE2 , iNOS
Proteins, lipids, and DNA
damage
TNF-, IL-1, IL-8
Exercise
MMP-9, ICAM-1, VCAM-1, cPLA2 Aging
Figure 1: Oxidative stress and inflammation overlapping sig- Figure 2: Modulation of oxidative stress and inflammation in aging
naling pathways in aging. AP-1 = activator protein-1, COX-2 = by exercise.
cyclooxygenase-2, cPLA2 = cytosolic phospholipase A2, ERKs =
extracellular signal regulated kinases, ICAM-1 = intercellular adhe-
sion molecule-1, IL-1 = interleukin-1, IL-8 = interleukin-8, iNOS =
inducible nitric oxide synthase, JNKs = c-jun N-terminal kinases, trapped within fat depots are able to release proinflammatory
LPO = lipoxygenase, MAPK p38 = mitogen activated protein kinase cytokines such as IL-6 and TNF- [148150, 181, 182]. Physical
p38, PI3K = phosphatidylinositol-4,5-bisphosphate 3-kinase, MMP- activity and exercise training increase energy expenditure
9 = matrix metalloproteinase-9, MPO = myeloperoxidase, NF- and reduce body fat, particularly visceral fat, with/without
B = nuclear factor kappa B, PGE2 = prostaglandin E2 , PKC = weight loss [35, 183, 184], and therefore reduced production
protein kinase C, RONS = reactive oxygen nitrogen species, Sph1P
and release of IL-6 and TNF- [185189]. Exercise training
= sphingosine-1-phosphate, TNF- = tumor necrosis factor-alpha,
VCAM-1 = vascular cell adhesion molecule-1, and XO = xanthine increased gene expression of PGC-1 alpha, a master regulator
oxidase. of mitochondrial biogenesis, in rat adipose tissue [190],
leading to increased energy expenditure particularly in the
visceral area. Exercise training inhibited the infiltration of
transcription factors such as NF-B and AP-1 resulting in the inflammatory phenotype M1 macrophages into adipose
the generation of large quantities of these proinflamma- tissue, while also favoring the switch of macrophages to
tory mediators and ROS (Figure 1). Indeed, aging is asso- the less inflammatory phenotype M2 in obese mice [191].
ciated with adverse health conditions such as atherosclero- Exercise training/physical activity also induces the release
sis, metabolic syndrome, sarcopenia, arthritis, and chronic of adiponectin from adipose tissues [192197]; adiponectin
obstructive pulmonary disease that are characterized by exerts antiapoptotic, anti-inflammatory, and antioxidative
elevated levels of both oxidative stress and inflammatory activities [198, 199].
markers [178].
Not surprisingly, ROS can also induce proteins such
5.2. Effect of Exercise Training on Skeletal Muscles. Physical
as heat shock proteins (HSPs), HSP70 in particular [179],
activity/exercise increases nutritive blood supply to and
and heme oxygenase 1 (HO-1) [180] that can protect cells
removes waste from skeletal muscles, while also upregulating
and tissues from the deleterious effects of inflammation.
the expression of the anabolic myokine IL-15 [195197, 200,
However, the balance of antioxidant/anti-inflammatory to
201]. Most importantly, physical activity/exercise stimulates
oxidant/inflammatory proteins is tilted towards the latter
mitochondrial biogenesis [202] and oxidative capacity [203]
during the aging process.
that provide energy for the synthesis of new proteins. Thus,
physical activity/exercise improves muscle mass and strength
5. Exercise Training: Modulation of Oxidative and renders them less vulnerable to acute injury [204],
Stress and Inflammation therefore suppressing triggers of inflammation and oxidative
damage [205209].
Exercise and regular physical activity counteract the dele- Physical activity/exercise also induces the release of
terious effects of aging, not only by combating sarcopenia, several myokines from skeletal muscle such as IL-6 [210
obesity, and mitochondrial dysfunction, the major triggers 212], which suppresses IL-1 and TNF- [213] and triggers
of oxidative stress and inflammation in aging, but also the release of many anti-inflammatory cytokines such as
by exerting additional antioxidant and anti-inflammatory IL-1 receptor antagonist (IL-1ra) and IL-10, in addition to
actions as illustrated in Figure 2. cortisol [214, 215]. In turn, IL-10 inhibits the synthesis of some
proinflammatory cytokines such as TNF- and IL-1 [200].
5.1. Effect of Exercise Training on Adiposity. Adipose tis- Exercise also reduces TNF- and IL-1 production in skeletal
sue, particularly visceral fat depots, and the macrophages muscles [212, 216, 217].
Heat shock proteins (HSPs) are also generated in skeletal intensity (mild/moderate/intense/exhaustive), and frequency
muscles in response to physical activity/exercise; they exert (sessions per day/week/month) of exercise and also on the
vital anti-inflammatory action as will be explained later [218 subjects characteristic (age, sex, endurance capacity, and
222]. health condition).
5.3. Effect of Exercise Training on Mitochondrial Aging. 5.4.1. Molecular Transducer of the Anti-Inflammatory Effects
Exercise mitigates mitochondrial aging and interrupts the of Exercise Training. The signaling pathways underlying the
vicious cycle of oxidative damage by stimulating mitochon- anti-inflammatory effects of exercise are complex and not
drial biogenesis [202] and enhancing mitochondrial oxidative completely understood. In addition to the effects of exer-
capacity [203, 223, 224]. Excellent reviews on this topic are cise on adipose tissue, skeletal muscles, and mitochondrial
available [225, 226]. biogenesis mentioned above, exercise exerts additional anti-
inflammatory actions on the immune system, repair mecha-
5.4. Anti-Inflammatory Effects of Exercise Training. Acute nisms, and vasculature.
bouts of exercise cause transient damage to contracting
skeletal muscles, triggering an inflammatory response that Effects of Exercise on the Immune System. Regular exercise
increases the levels of proinflammatory cytokines and acute- downregulates the innate immune response and activates
phase reactants in the blood [227230]. However, regular the adaptive immune system with consequent suppression
exercise reduces levels of systemic inflammatory markers of inflammation. Exercise modulates the immune system
such as CRP, IL-6 TNF-, soluble TNF- receptor 1 (sTNF- by reducing the number of inflammatory CD14+CD16+
R1), and soluble TNF- receptor 2 (sTNF-R2) in young and monocytes [250], increasing the number of CD4CD25 regu-
middle aged adults [231243] and also more importantly latory T cells [264, 283], shifting blood macrophages towards
in the elderly [195, 244250]. Additionally, higher levels the less inflammatory phenotype M2 [284], increasing the
of the anti-inflammatory cytokines interleukin-10 (IL-10) dominance of the anti-inflammatory Type 2 helper T cell
[246] and adiponectin [195] are associated with increased over proinflammatory Type 1 helper T cell [265, 284286],
physical activity in the elderly. Several interventional studies and reducing monocyte chemoattractant protein-1 (MCP-1)
report that exercise reduces inflammatory markers, partic- [188] and toll-like receptor-4 (TLR4) expression on monocyte
ularly CRP, TNF-, interferon-gamma (INF-), monocyte surfaces [239, 263, 275]. On the other hand, ET increases
chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), the production of transforming growth factor beta (TGF1)
interleukin-18 (IL-18), sTNFR2, sTNF-R1, and soluble IL-6 [254, 264] from regulatory T cells.
receptor (sIL-6R), and increases levels of anti-inflammatory Exercise also stimulates the sympathetic nervous sys-
factors such as IL-10, interleukin-12 (IL-12), interleukin-4 (IL- tem and the hypothalamic-pituitary-adrenal axis to increase
4), and transforming growth factor beta 1 (TGF1) [74, 188, serum glucocorticoid levels [287] with subsequent inhibition
197, 217, 251265]. These benefits of exercise also occur in of the immune system [288].
the elderly [100, 196, 250, 263, 266271] as summarized in
Table 1. However, only a few randomized controlled trials Effects of Exercise on Repair Mechanisms. Heat shock proteins
confirm the anti-inflammatory effect of exercise [74, 261, 262, are highly conserved chaperone proteins that regulate the
269]. Exercise training can exert anti-inflammatory effects folding and processing of damaged proteins and therefore
with/without accompanied weight loss; however, the most exert significant anti-inflammatory action. Numerous studies
substantial anti-inflammatory effects occur in patients with have shown that exercise is capable of upregulating the
high baseline inflammatory biomarkers, particularly when expression of HSP72 [219, 222, 289293], HSP70 [137, 218,
associated with weight loss [260]. 220, 221, 294297], HSP60 [294, 298], HSP27 [294, 295],
It is worth noting that some interventional and random- and HSP25 [222] in skeletal muscles, blood cells, hearts, and
ized controlled trials studies did not detect a significant effect arteries of humans as well as young and aged experimental
of regular exercise on systemic inflammatory biomarkers in animals (see Table 2). However, Hagg et al. [299] reported
adults [243, 272274] or in aged adults [275278] as shown that voluntary wheel running of spontaneously hypertensive
in Table 1. A meta-analysis found only five randomized rats for 5 weeks reduced aortic gene expression of HSP70
controlled trials that examined the effects of regular aerobic and HSP60. The effect of exercise on HSPs depends on age
exercise (of at least 4-week duration) in adults and concluded [296], sex [220], time course [221, 291], and HSP subtype
that aerobic exercise did not reduce CRP levels [279]. It is [222, 293, 294, 298].
likely that these discrepancies may be attributed to the smaller
sample size used in the clinical trials examined. Effects of Exercise on Vascular Endothelial Cells. By increasing
On the other hand, the effects of resistance exercise shear stress on vascular endothelial cells, exercise modulates
on inflammatory mediators are mostly negative [280282], key players in the inflammatory process such as ICAM-
although Brooks et al. [196] reported that 16 weeks of 1, NF-B, MAPK, and COX-2 [300, 301]. Voluntary wheel
resistance training reduced CRP and increased adiponectin running of aged mice for 1014 weeks reduced the activation
levels in older diabetic patients. The effects of physical of NF-B in the aorta [302]. Subjecting aortic endothelial
activity and different exercise programs on inflammatory cells to in vitro shear stress for 4 h reduced the expression of
mediators in the elderly are detailed in Table 1. Clearly, the vascular cell adhesion molecule-1 (VCAM-1) [303]. Treadmill
effects of exercise depend on the type (aerobic/resistance), training for 13 weeks reduced the expression of intercellular
Table 1: Effect of physical activity/exercise on inflammatory mediators in the elderly.
Study Mediator Subjects Tissue Physical activity/exercise Effect of physical activity/exercise Reference
65 years Inverse association between log TNF-
Observational TNF- Plasma Self-reported physical activity [249]
= 1004 and physical activity
6580 years Lower percentage in the physically
Serum Regular exercise [250]
= 30 active subgroup
65 years Inverse association between physical
CRP Blood Self-reported physical activity [241]
= 5,888 activity and CRP
Men, 58 years Self-reported leisure time physical Inverse association between physical
Blood [242]
= 391 activity activity and CRP
70 to 79 years Inverse association between CRP and
Blood Self-reported physical activity [244]
= 870 physical activity
70 to 79 years Previous week exercise and physical Inverse association between physical
Blood [245]
activities activity and CRP
= 3,075
Plasma Self-reported physical activity [247]
Physical function measures included
Plasma handgrip strength, signature time, [248]
= 880 higher walking speed and grip strength
chair stands, and 6-minute walk time
65 years Inverse association between and log
= 1004 CRP and physical activity
6580 years Lower level in the physically active
Serum Regular exercise [250]
= 30 subgroup
Men,
Lower levels of IL-6 in the physically
IL-6 6574 years Serum Self-reported physical activity [246]
active group
= 12
70 to 79 years Previous week exercise and physical Lower level associated with higher
Blood [245]
= 3,075 activities level of physical activity
70 to 79 years Inverse association between IL-6 and
Blood Self-reported physical activities [244]
65 years Inverse association between log IL-6
= 1004 and physical activity
Physical function measures included
70 to 79 years Inverse association between IL-6 and
Plasma handgrip strength, signature time, [248]
= 880 higher walking speed
chair stands, and 6-minute walk time
Men,
Higher levels of IL-10 in the physically
IL-10 6574 years Serum Self-reported physical activity [246]
active group
= 12
6580 years Lower percentage in the physically
CD14+CD16+ Serum Regular exercise [250]
= 30 active subgroup
50 to 70 years Direct association between
Adiponectin Plasma Self-reported physical activity [195]
= 3289 adiponectin and physical activity
5
6
Table 1: Continued.
64 years Aerobic or flexibility/strength exercise Reduced level by aerobic and strength
Interventional TNF- Blood [266]
= 105 for 10 months exercise
Men, 67 8 years with
congestive heart failure Plasma Exercise training for 3 months Level reduced after training [267]
= 28
81 1 years Skeletal Reduced mRNA and protein levels
Exercise training for 3 months [268]
= 13 muscle after training
6580 years,
3 days/week endurance and resistance Reduced level compared with
physically inactive Blood [250]
exercise training for 12 weeks pretraining values
= 15
Postmenopausal women,
6580 years Blood Regular exercise for previous 6 months No change in protein or mRNA [275]
= 20
6580 years Progressive resistance strength
Serum No change [280]
=8 training for 12 weeks
Overweight/obese sedentary
Combined weight training and
with knee osteoarthritis
sTNF-R1 Serum walking for 1 h, 3 times/week for No change [277]
60 years
18 months
= 316
Type 2 diabetic patients,
CRP >55 years Serum Strength training for 16 weeks Reduced level after training [196]
= 62
>64 years Aerobic or flexibility/strength exercise Reduced level by aerobic but not
Blood [266]
= 105 for 10 months strength exercise
Postmenopausal overweight
or obese, sedentary women, Moderate-intensity aerobic exercise for
Serum Level reduced after training [269]
5075 years 12 months
= 115
Women with the metabolic
Four sessions of high-intensity aerobic
syndrome,
Blood and resistance exercise per week for Level reduced after training [271]
68.7 3.4 years
12 months
= 32
Patients with CHD, 66.7
11 years
Cardiac rehabilitation and exercise
= 235 Blood Level reduced after training [100]
training for 3 months
Controls 63.9 11.1 years
= 42
60 to 85 years
Serum Exercise training for 6 months No change [276]
= 30
Serum walking for 1 h, 3 times/week for No change [277]
60 years
18 months
= 316
Table 1: Continued.
Postmenopausal breast cancer
survivors,
Serum Cycling 3 times/week for 15 weeks No change [278]
50 to 69 years
= 52
IL-6 Blood [266]
Moderate-intensity combination of
7089 years
Plasma aerobic, strength, balance, and Reduced IL-6 level but not CRP [270]
= 424
flexibility exercises for 12 months
Young (2030 years) and aged Endurance (20 min) and resistance Stimulated level was reduced in young
Blood [263]
(6676 years) = 60 exercise 3 days/week for 12 weeks and old subjects
6580 years Blood Regular exercise for 6 months No change in protein or mRNA [275]
= 20
Serum walking for 1 h, 3 times/week for No change [277]
60 years
18 months
= 316
Serum No change [280]
IL-1 Serum No change [280]
IL-18 Blood [266]
TLR4 Blood Regular exercise for 6 months Lower level in trained versus untrained [275]
6580 years
Young (2030 years) and aged
Endurance (20 min) and resistance Level reduced in young and old
(6676 years) CD14+ cell [263]
exercise 3 days/week for 12 weeks subjects
= 60
6580 years sedentary Endurance and resistance exercise Reduced level compared with
CD14+CD16+ Blood [250]
= 15 training for 12 weeks (3 days/week) pretraining values
Type 2 diabetic patients
Adiponectin >55 years Serum Strength training for 16 weeks Increased level after training [196]
= 62
7
8
Table 2: Effects of exercise training on HSPs in humans and experimental animals.
HSP Species Tissue Physical activity/exercise mode Effect of physical activity/exercise References
Men,
HSP72 Human 22.1 3.8 years Plasma Semirecumbent cycling for 120 min Levels increased after exercise [289]
=7
Men and women,
Protein expression increased during
2230 years Serum Acute bout of treadmill running for 60 min [219]
and after exercise
=6
Men and women,
Skeletal
2230 years Acute bout of treadmill running for 60 min mRNA level increased after exercise [219]
muscle
=6
Young (3 months) and aged Skeletal Protein expression increased in
Rats 4.5 weeks of resistance exercise [222]
(30 months) muscle young and old rats
1 or 3 consecutive days for 100 min at a speed
Adult females Heart Increased expression [290]
of 20 m/min
Adult males Heart Treadmill running for 1 or 3 days Increased levels after 3 but not 1 day [291]
Adult males Heart 24-week but not 12-week treadmill training Increased expression [292]
Females,
Heart Endurance exercise for 10 weeks Increased expression [293]
4 months
Females, 35 consecutive days of treadmill exercise
Ventricle Increased levels [368]
4 months [60 min/day at 6070% maximal O2 uptake]
Male athletes,
HSP70 Human 32.3 9.3 years Leukocytes Half marathon run Protein expression increased [294]
= 12
Men and women, mRNA level but not protein level
Skeletal
26 4 years 30 min on a treadmill increased at 4 min, 30 min, and 3 h [221]
muscle
=5 after exercise
Women,
Skeletal Protein expression increased after
22 2.2 years Acute bout of eccentric contractions [218]
muscles exercise
= 10
Treadmill training for 30 m/min,
Rats Aged 24 months old Hearts Expression increased [295]
45 min/day, 5 days/week for 6 weeks
Protein increased in the young
Young (6 months) and aged Treadmill for 60 min/day, 5 days/week for a
Left ventricle group compared with sedentary [296]
27 months total of 12 weeks
control
Males, young (4 months) and Acute exercise for 60 min at 7075% of Expression increased in young and
Heart [297]
aged (21 months) maximum oxygen consumption old rats
Males,
Heart Treadmill for 3 days/week for 14 weeks Increased protein level [137]
2 months
Spontaneously hypertensive
Aorta Voluntary wheel running for 5 weeks Reduced gene expression [299]
females (9 weeks)
Males and females, Skeletal Protein and mRNA expression
Acute treadmill running for 30 min [220]
11 weeks muscle increased in males but not females
Males Cardiac
Mice Swimming training for 14 weeks No change [298]
(68 weeks) ventricles
Table 2: Continued.
HSP Species Tissue Physical activity/exercise mode Effect of physical activity/exercise References
Male athletes,
HSP60 Human 32.3 9.3 years Leukocytes Half marathon run Expression increased [294]
= 12
Males,
Rats Heart Treadmill for 3 days/week for 14 weeks Decreased mRNA [137]
2 months
Spontaneously hypertensive
Aorta Voluntary wheel running for 5 weeks Reduced gene expression [299]
females (9 weeks)
Males,
Mice Ventricles Swimming training for 14 weeks Increased level [298]
68 weeks
Females,
HSP32 Rats Heart Endurance exercise for 10 weeks No change [293]
4 months
Male athletes,
HSP27 Human 32.3 9.3 years Leukocytes Half marathon run Expression increased [294]
= 12
Treadmill training for 30 m/min,
Rats Males, aged (24 months) Hearts Expression increased [295]
Males, young (3 months) and Skeletal Protein expression increased in
HSP25 Rats 4.5 weeks of resistance exercise [222]
aged (30 months) muscles young and old rats
Male athletes,
HSC70 Human 32.3 9.3 years Leukocytes Half marathon run No change [294]
= 12
Males, young (3 months) and Skeletal
Rats Resistance exercise for 4.5 weeks No change in protein expression [222]
aged (30 months) muscles
9
Exercise
Immune cells Skeletal muscles Adipose tissue Vasculature

CD14+CD16+ mitochondrial biogenesis visceral fat shear stress
TLR4 muscle mass and strength IL-6 NF-B
INF, TNF-, IL-1 IL-15 TNF- VCAM-1
IL-8, MCP-1 HSPs macrophages ICAM-1
sIL-6R, sTNFR2 IL-6 IL-10, IL-1ra infiltration HSPs
CD4CD25 Treg TNF-, IL-1 M1 : M2
phenotype
Th2 : Th1
adiponectin
M2 : M1
IL-10, IL-4
TGF1, adiponectin
HSPs
Figure 3: Signaling pathways underlying the anti-inflammatory actions of exercise. HSPs = heat shock proteins, IL-1 = interleukin-1-alpha,
IL-1ra = interleukin-1 receptor antagonist, IL-1 = interleukin-1 beta, IL-6 = interleukin-6, IL-8 = interleukin-8, IL-10 = interleukin-10, IL-15
= interlukin-15, INF = interferon gamma, M1 = macrophage phenotype 1, M2 = macrophage phenotype 2, ROS = reactive oxygen species,
sTNFR2 = soluble TNF- receptor 2, sIL-6R = soluble IL-6 receptor, TLR4 = toll-like receptor-4, TGF1 = transforming growth factor beta
1, TNF- = tumor necrosis factor-alpha, Th1 = Type 1 helper T cell, and Th2 = Type 2 helper T cell.
cell adhesion molecule-1 (ICAM-1) in response to cerebral condition was markedly improved. Another clinical study
ischemia in rats [304]. The mechanisms underlying the anti- showed that 8 weeks of walking exercise did not significantly
inflammatory actions of exercise are summarized in Figure 3. change low density lipoprotein (LDL) oxidation or nitration
in the elderly [320].
5.5. Antioxidant Effects of Exercise Training. Generation
of ROS is transiently increased during exercise; however, 5.5.1. Molecular Transducer for the Antioxidant Effects of Exer-
the incidence of diseases associated with oxidative stress cise Training. As discussed above, exercise exerts prominent
is reduced by regular exercise. Regular exercise attenuates anti-inflammatory actions, thus suppresses major sources of
oxidative damage in the brain [23, 305307], liver [23, 130, ROS and RNS generation, and produces indirect antiox-
308310], kidney [23, 136], skeletal muscle [311], blood [113, idant effects. Exercise also upregulates the antioxidant
136], and heart [23, 297]. However, Goto et al. [135] found defense mechanisms and repair proteins in the body via
that high-intensity exercise for 12 weeks increased the indices redox-sensitive transcription factors, mainly NF-B, activa-
of oxidative stress in young men. tor protein-1 (AP-1) and peroxisome proliferator-activated
Importantly, regular exercise ameliorates age-associated receptor gamma coactivator 1-alpha (PGC-1), and by
oxidative stress in the heart [297, 312], liver [130], plasma increasing laminar shear stress on vascular endothelial cells.
[113], arteries [138], and skeletal muscles [313, 314]. In the The metabolic demands of skeletal muscles increase dur-
study of Navarro et al. [23], exercise reduced age-associated ing exercise; the body responds by increasing oxygen uptake
mitochondrial oxidative damage and upregulated mitochon- and blood flow to the muscles and other body organs. The
drial NADH-cytochrome C reductase and cytochrome oxi- increased metabolic rate results in greater ROS production
dase activities in brain, heart, liver, and kidney of 52-week- in skeletal muscles [128, 321] and in other organs as well [129,
old but not older rats. However, exercise caused an increase 322]. Sources other than the electron transport chain enzymes
in oxidative damage in skeletal muscles [315] and hearts of in the mitochondria, such as xanthine oxidase [323325] and
aged rats [316]. NADPH oxidase [128, 326], contribute to ROS generation
In elderly people, regular exercise reduced serum/plasma during exercise. This transient increase in ROS levels activates
levels of myeloperoxidase, a marker of inflammation and NF-B, AP-1, and PGC-1 signaling.
oxidative stress [205], and thiobarbituric-reactive acid sub-
stances, a marker of lipid peroxidation [317]. Lower levels Effects of Exercise on NF-B and AP-1 Signaling. Exercise-
of nitrotyrosine [133] and thiobarbituric-reactive acid sub- induced increase in ROS levels triggers an adaptive antiox-
stances [318] were found in the more physically active elderly idant response that is mediated via mitogen activated protein
people. However, de Gonzalo-Calvo et al. [319] reported that kinases (MAPK p38, ERK 1, and ERK 2) [323, 327329],
although regular exercise increased protein carbonyl content cAMP-response-element binding (CREB) [330, 331], and
and lipid peroxidation levels in the plasma and erythro- synapsin [330, 331], to activate redox-sensitive transcription
cytes of long term trained elderly men, their overall health factors such as NF-B [323, 332, 333] and AP-1 [327, 333],
resulting in increased expression of antioxidant enzymes muscles [136, 311, 358], and arteries [359, 360] of experimental
[334] such as superoxide dismutase (SOD) [323, 333] and animals, particularly of aged animals [311, 316, 356, 358, 360].
catalase [333], repair proteins such as hear shock proteins
HSP25, HSP60, HSP72, HSP70, and heat shock cognate 70 (1) SOD-1. Just increasing in vitro shear stress is sufficient
HSC70 [315, 333336], proteasomes complex, and nitric oxide to upregulate the gene and protein expression of SOD-1 in
synthase (NOS) [308, 323]. These signaling cascades were human endothelial cells [349351]. A study by Ennezat et
demonstrated in skeletal muscles [323, 332], brain [330, 331], al. [361] showed that regular exercise for 12 weeks increased
leukocytes [337], and hearts [327] of experimental animals as gene expression of SOD-1 in skeletal muscles of congestive
well as in humans [328, 337] and in aged animals [333, 335, heart failure patients. Increases in protein expression of SOD-
338] and elderly people [337]. However, other studies report 1 were observed after exercise in skeletal muscles [222, 362],
that exercise-induced activation of NF-B and AP-1 [333] heart [295, 363], brain [307], and arteries [194, 364, 365] of
and upregulation of HSP70 were attenuated in fast skeletal several experimental animals, importantly of aged animals
muscles of old rats [339]. Interestingly, aging also increased [295]. However, other studies reported decreased or no
ROS production and NF-B activity in the livers of aged rats; change in expression of SOD-1 following long term exercise
these effects were attenuated by exercise [130, 308]. in aged animals [222, 366] and adult animals [367].
Effects of Exercise on PGC-1 Signaling. Exercise stimulates (2) SOD-2. Increases in activity have been observed in heart
mitochondrial biogenesis [202] and ameliorates the age- [230, 292, 296, 297, 368370], skeletal muscles [371], plasma
associated decline in mitochondrial oxidative capacity in [137], and liver [372] of experimental animals following
skeletal muscles [223] and other organs [23, 190, 340] via exercise. Increased protein expression has been reported in
PGC-1 signaling [190, 341, 342]. PGC-1 is a redox-sensitive plasma and vascular endothelial cells of humans [133, 373]
transcription factor that is activated by 5 -AMP-activated and in heart [230, 295, 296, 363, 374], skeletal muscles [362,
protein kinase (AMPK) [329, 343345] to trigger the tran- 371, 375], liver [372], and arteries [138, 376] of experimental
scription of nuclear respiratory factor 1 (NRF-1) and expres- animals, notably in aged animals [138, 295, 296, 375].
sion of mitochondrial transcription factor A (mtTFA), a key
regulator of mitochondrial DNA replication [346]. PGC-1 (3) SOD-3. Higher activity was found in more physically
also increases the expression of antioxidant proteins such as active older men [133] and increased protein expression
glutathione peroxidase (GPX) and SOD-2 [347]. Safdar et al. was observed in men after a bout of acute exercise but
[348] report that exercise reversed most of the multisystem not endurance training [373]. However, increased protein
pathology and premature mortality in mice which were levels were detected in experimental animals after endurance
genetically modified to accumulate mitochondrial mutations. exercise [366, 367].
The effects of exercise on AMPK and PGC-1 were preserved
in the hippocampus of aging rats. However, results from (4) Catalase. Winter swimmers had higher catalase activity in
Derbre et al. [341] suggest a blunted effect of exercise response their erythrocytes than untrained subjects [353], while sprint-
in PGC-1 and NRF-1 in skeletal muscles of aged rats. trained athletes exhibited lower activity than controls [352].
Exercise increased catalase activity in brain [23, 377, 378],
Effects of Exercise on Vascular Endothelial Cells. To meet the heart [23, 136, 297, 316, 370], lung [377], skeletal muscles
increasing metabolic demands of the body during exercise, [136, 371, 377], liver [23, 126, 136, 356, 377], kidney [23],
perfusion of skeletal muscles and other tissues increases, sub- and cardiac mitochondria [379] of experimental animals and
jecting vascular endothelial cells to higher levels of laminar importantly in aged animals [126, 297, 316, 356, 377, 378].
shear stress. Increased laminar shear stress modulates gene However, other studies reported reduced [292, 311, 380] or no
expression and activity of SOD [349351] possibly via NF-B change in catalase activity after endurance exercise [293, 365].
and MAPK signaling [300, 301].
(5) GPx. Athletes had higher activity than untrained subjects
Effects of Exercise on Antioxidant and Prooxidant Enzymes
[352]. Also, physically active elders had higher activity than
Expression and Activity. The NF-B, AP-1, PGC-1, and shear
less active individuals in their erythrocytes [381]. Long
stress signaling cascades converge to upregulate antioxidant
defense mechanisms to counteract and interrupt the vicious term endurance exercise increased GPx activity in healthy
cycle of inflammation and oxidative stress associated with adults [354, 355] and upregulated gene expression in con-
aging and cardiovascular diseases. The most intensely studied gestive heart failure patients [361]. In experimental animals,
antioxidant enzymes in laboratory animals and in humans increased activity was observed in heart [136, 356, 382, 383],
are SOD, catalase, GPx, glutathione transferase (GST), and liver [126, 136, 356, 372, 377, 382], lung [356, 377], kidney
glutathione reductase (GSR). The effects of exercise on [136], brain [377, 378], testes [377], and skeletal muscles
antioxidant enzymes are summarized in Table 3. [311, 313, 371, 382384], particularly in aged animals [126, 313,
Athletes erythrocytes had higher SOD activity compared 356, 377, 378, 384]. Other investigators reported reduction
with untrained individuals [352, 353]. Regular and acute [380] and no change [292, 293, 298] in GPx activity fol-
exercise increased SOD activity in erythrocytes of men and lowing endurance exercise. Increased GPx gene and protein
women [354, 355] and heart [23, 136, 292, 293, 316, 356 expression following long term endurance exercise were also
358], lung [23, 356], kidney [23, 136], brain [23], skeletal reported [307, 372].
12
Table 3: Effects of exercise training on expression and activity of antioxidant and prooxidant enzymes.
Enzyme Species Tissue Exercise mode Effect of physical activity/exercise Reference
Untrained males High-intensity endurance training for
SOD Human Erythrocytes Activity increased after training [354]
=9 12 weeks
Healthy young men and
16 weeks of training then an acute bout of Transient increase in activity after
women Erythrocytes [355]
aerobic exercise for 30 min acute exercise
= 17
Athletes = 18 and sedentary Higher activity in sprint-trained
Erythrocyte Marathon or sprint training [352]
control = 6 athletes and marathon runners
Winter swimmers = 40 and
Erythrocyte Regular winter swimming Higher activity in winter swimmers [353]
controls = 36
Males, young and aged Increased activity in lung and heart of
Rats Lung, heart, and liver Regular swimming exercise for 1 year [356]
(17 months) old rats relative to sedentary controls
Increased activity in young and aged
Males, young and aged Heart Treadmill endurance exercise for 2 months [316]
rats
Increased activity in deep vastus
Male, young, adult, and aged Skeletal muscles Exercise training for 10 weeks [311]
lateralis muscle of young rats only
Males, 16-17 weeks Heart and skeletal muscle Sprint training on a treadmill for 6 weeks Unchanged activity [383]
Females, 4 months Ventricles Endurance exercise training for 10 weeks Increased activity [293]
High-intensity exercise treadmill for
Females, 17 weeks Ventricles Increased activity [357]
10 weeks
Increased activity after 24 but not
Males, adults Heart Treadmill training for 12 or 24 weeks [292]
12 weeks
Treadmill training 5 times per week,
Males, myocardial infarcted Aorta Increased activity but not expression [359]
60 min/day for 11 weeks
Mice Males, aged 2932 months Aorta Voluntary wheel running for 1014 weeks Increased activity [360]
Males and females, aged 28, Bain, heart, liver, and Long term moderate-intensity treadmill Increased activity in all tissues at 52
[23]
52, and 78 weeks kidney exercise but not 78 weeks old
Kidney, heart, and skeletal
Females, 3 months Treadmill exercise for 8 weeks Increased activity [136]
muscle
Males
Ventricles Swimming training for 14 weeks No change in activity [298]
(68 weeks)
Short-tailed field vole
Microtus Skeletal muscle and heart Voluntary running over 1 or 7 days Reduced activity in the heart [401]
Microtus agrestis
Table 3: Continued.
Umbilical vein endothelial mRNA and protein levels increased
SOD-1 Human Laminar fluid shear stress [350]
cells after 24 hours
Increased mRNA expression and
Endothelial progenitor cells Shear stress [349]
activity
Aortic endothelial cells Fluid shear stress Increased protein expression [351]
Patients with congestive heart

Skeletal muscle 12 weeks of training Increased gene expression [361]
failure = 14
Young (3 months) and aged Protein expression increased in young
Rats Skeletal muscles 4.5 weeks of resistance exercise [222]
(30 months) rats but decreased in old rats
Increased activity in skeletal muscles
Males, young (35 months)
Skeletal muscles and heart Exhausting treadmill running and heart of young rats and hearts of [358]
and aged (2427 months)
old rats
Treadmill training 30 m/min, 45 min/day, 5
Males, aged 24 months Hearts Protein expression increased [295]
days/week for 6 weeks
Males, young (2 months) and
Soleus muscle feed arteries Exercise training for 1012 weeks No change in protein expression [366]
old (22 months)
Females,
Hippocampus Treadmill training for 15 weeks Protein expression increased [307]
12 months old
Increased protein level but not mRNA
Females Skeletal muscle Acute bout of exhaustive treadmill exercise [362]
or activity
Acute session of treadmill running for
Males, adults Heart No change [230]
2530 min
Females, adults Ventricles 20 weeks of training Increased protein expression [363]
Aorta and mesenteric Increased expression relative to
Males, high caloric fed Running 60 min, 5 days/week for 12 weeks [364]
artery sedentary controls
Treadmill running 15 m/min, 30 min/day, 5
Mice Aorta No change in protein expression [367]
Males, diabetic young Aorta Treadmill exercise for 10 weeks Increased protein expression [194]
Pigs Females Aortic endothelial cells Chronic exercise training for 1619 weeks Protein and activity increased [365]
13
14
Table 3: Continued.
Men Swimming or running for 3 months then a Protein level increased by acute
SOD-2 Human Plasma [373]
= 18 bout of acute exercise exercise
Men 62 3 years
Vascular endothelial cells Higher protein expression than
Physically active = 13 and Habitual aerobic exercise [133]
from the brachial artery sedentary men
sedentary = 26
Rats Males, 10-11 weeks Cardiac mitochondria Long term voluntary wheel running Reduced activity [402]
Increased activity after 24 but not
12 weeks
Activity increased at 0.5 and 48 h, and
Acute session of treadmill running for
Males, adults Heart protein content increased at 48 h after [230]
2530 min
exercise
Females, 35 consecutive days of treadmill exercise
Heart Increased activity [368]
4 months [60 min/day at 6070% maximal O2 uptake]
Treadmill exercise (60 min/day) at 25
Females, 4 months Ventricles Increased activity [369]
degrees for 3 days
Females, adults Ventricles 20 weeks of training Increased protein expression [363]
3 consecutive days of intensive treadmill Increased activity of SOD-2 but not
Males subjected to IR Ventricles [370]
exercise 60 min/day, at 30 m/min SOD-1
Treadmill training 30 m/min,
Males, aged 24 months Heart Protein expression increased [295]
Males, young (4 months) and Acute exercise 60 min at 7075% of
Heart Activity increased in old rats [297]
aged (21 months) maximum oxygen consumption
Protein expression and activity
Young (6 months) and aged Treadmill for 60 min/day, 5 days/week for a
Left ventricle increased in the aged group compared [296]
27 months total of 12 weeks
with sedentary control
Increased activity and protein
Females Skeletal muscle Treadmill running for 10 weeks [371]
expression
Increased mRNA level in deep vastus
Females Skeletal muscles Acute bout of exhaustive treadmill exercise lateralis muscle. Increased protein level [362]
in superficial vastus lateralis
Male Zucker diabetic fatty rats
Skeletal muscles Swimming training for 6 weeks Protein expression increased [375]
(18 weeks)
Males,
Plasma Treadmill training 3 days/week for 14 weeks Increased activity [137]
2 months
Treadmill running at 20 m/min for 1 h/day, 7 mRNA and protein levels and activity
Males, obese Zucker Liver [372]
days/week, for 8 weeks increased
Male, young (3 months) and Increased protein expression in aged
Aorta Treadmill training for 12 weeks [138]
aged (23 months) rats
Motorized exercise-wheel for 1 h/day, 5
Mice Male, diabetic and young Heart Increased protein expression [374]
Motorized exercise-wheel for 1 h/day, 5
Male, diabetic and young Aorta Increased protein expression [376]
Pigs Females Aortic endothelial cells Chronic exercise training for 1619 weeks No change in protein levels [365]
Table 3: Continued.
Men 62 3 years
Vascular endothelial cells
SOD-3 Human Physically active = 13 and Habitual aerobic exercise Higher activity than sedentary men [133]
from the brachial artery
sedentary = 26
Men Swimming or running for 3 months then a Reduced protein level after endurance
Plasma [373]
= 18 bout of acute exercise training but increased by acute exercise
Rats Soleus muscle feed arteries Exercise training for 1012 weeks Increased protein expression in old rats [366]
old (22 months)
Treadmill running 15 m/min, 30 min/day, 5
Mice Aorta Increased protein expression [367]
days/ week for 3 weeks
Athletes = 18 and sedentary Lower activity than controls in
CAT Human Erythrocytes Marathon or sprint training [352]
control = 6 sprint-trained athletes
Winter swimmers = 40 and
Erythrocytes Regular winter swimming Higher activity in winter swimmers [353]
controls = 36
Males, young and aged Increased activity in liver of old rats

Rats Lung, heart and liver Regular swimming exercise for 1 year [356]
(17 months) relative to sedentary controls
Brain, liver, lung, muscle,
Aged Regular exercise Increased activity in all tissues [377]
and testes
Males, young (4 months) and Acute exercise 60 min at 7075% of Activity increased in young and old
Heart [297]
aged (21 months) maximum oxygen consumption rats
Increased activity in young and aged
Male, young and aged Heart Treadmill exercise for 2 months [316]
rats
Liver Regular exercise Increased activity [126]
aged (20 months)
Male, young (8 months) and Swimming 30 min/day, 5 days/week for Increased activity in hippocampus in
Brain [378]
aged (22 months) 12 weeks young and old rats
Male, young (8 weeks), adult
Decreased activity in soleus muscle of
(12 months), and old Skeletal muscles Exercise training for 10 weeks [311]
adult and old rats
(24 months)
Treadmill for 16 weeks (5 days/week,
Males (4 months) Cardiac mitochondria Increased activity [379]
60 min/day, 25 m/min)
Reduced activity after 24 but not
12 weeks
Females, 4 months Ventricles Endurance exercise training for 10 weeks No change in activity [293]
3 consecutive days of intensive treadmill
Males subjected to IR Ventricles Increased activity [370]
exercise 60 min/day, at 30 m/min
lateralis muscle
Male, normotensive and Liver, kidney, skeletal Reduced activity in all tissues in
Treadmill running for 10 weeks [380]
hypertensive (11-12 weeks) muscles, and heart hypertensive and normotensive rats
Males and females, aged 28, Brain, heart, liver, and Long term moderate-intensity treadmill Increased activity in all issues at 52-
Mice [23]
52, and 78 weeks kidney exercise but not 78-week-old mice
Liver, heart, skeletal
Females, 3 months Treadmill for a total of 8 weeks Increased activity [136]
muscle, and salivary gland
Pigs Females Aortic endothelial cells Exercise training for 1619 weeks No change in protein level [365]
15
16
Table 3: Continued.
Untrained males High-intensity endurance training for
GPX Human Erythrocytes Increased activity after training [354]
=9 12 weeks
Activity increased after regular
Healthy young 16 weeks of training then an acute bout of
Blood training and transiently reduced [355]
= 17 aerobic exercise for 30 min
following acute exercise
Exercising and sedentary,
Higher activity in the exercising elderly
young (2138 years) and old Erythrocyte Regular exercise [381]
compared to the sedentary elderly
(6575 years) = 50
Athletes = 18 and sedentary Higher activity in sprint-trained
Erythrocyte Marathon or sprint training [352]
control = 6 athletes
Moderate-intensity semirecumbent bicycle
Patients with CHF = 14 Skeletal muscle Increased gene expression [361]
training for 12 weeks
Activity increased in liver, lung, and
Males, young and aged
Rats Lung, heart, and liver Regular swimming for 1 year heart of old rats relative to sedentary [356]
(17 months)
controls
Brain liver, lung, muscle, Increased activity in brain, liver, lung,
Aged Regular exercise [377]
and testes and testes
Male, young (8 months) and Swimming 30 min/day, 5 days/week for Increased activity in hippocampus in
Brain [378]
aged (22 months) 12 weeks young and aged
Females,
Hippocampus Treadmill for a period of 15 weeks Protein expression increased [307]
12 months old
Females Treadmill training (60 min, 5 days/week for
Skeletal muscles Increased activity [313]
(24 months) 10 weeks)
Male, young (8 weeks), adult
lateralis muscle of young rats only
(24 months)
Young (5 months) and aged Increased activity in deep vastus
Skeletal muscles Treadmill training for 10 weeks [384]
(27.5 months) lateralis muscle of old rats only
liver Regular exercise Increased activity [126]
aged (20 months)
Treadmill running at 20 m/min for 1 h/day, 7 mRNA and protein levels and activity
Males, obese Zucker Liver [372]
days/week for 8 weeks increased
Males, young Liver, heart, and muscle Swim training for 10 weeks Increased activity in all tissues [382]
Females, 4 months Ventricles Endurance training for 10 weeks No change in activity [293]
Males, adult Heart Treadmill training for 12 or 24 weeks No change in activity [292]
Increased activity in heart and some
Males, 16-17 weeks Skeletal muscle and heart Sprint training on a treadmill for 6 weeks [383]
skeletal muscle fibres
lateralis muscle
Male, normotensive and Liver, kidney, skeletal Reduced activity in all tissues in
Treadmill running for 10 weeks [380]
hypertensive (11-12 weeks) muscles, and heart hypertensive and normotensive rats
Mice Females, 3 months Liver, kidney, and heart Treadmill for a total of 8 weeks Increased activity [136]
Males
Ventricles Swimming training for 14 weeks No change in activity [298]
(68 weeks)
Table 3: Continued.
Healthy young 16 weeks of training then an acute bout of Activity increased after regular
GSR Human Plasma [355]
= 17 aerobic exercise for 30 min training
Males
Mice Ventricles Swimming training for 14 weeks No change in activity [298]
(68 weeks)
Rats Males, adults Heart Treadmill training for 12 or 24 weeks No change in activity [292]
Increased activity in heart and some
Males, 16-17 weeks Skeletal muscle and heart Sprint training on a treadmill for 6 weeks [383]
skeletal muscle fibres

Males, young Liver, heart, and muscle Swim training for 10 weeks Increased activity in all tissues [382]
Brain liver, lung, muscle,
Aged Regular exercise Increased activity in testes [377]
and testes
Activity increased in deep vastus
Males, young (8 weeks), adult
lateralis muscle of young rats and
decreased in soleus muscle of adult
(24 months)
rats only
GST Mice Females, 3 months Liver and salivary gland Treadmill for a total of 8 weeks Increased activity [136]
Patients with symptomatic
NAD(P)H Reduced protein and gene expression
Human coronary artery disease Internal mammary artery Aerobic training for 4 weeks [386]
oxidase and activity
= 45
Men, 62 3 years, physically
Vascular endothelial cells Lower level of p47(phox) compared
active = 13 and sedentary = Habitual aerobic exercise [133]
from the brachial artery with sedentary men
26
Males, young and myocardial Treadmill training 5 times per week,
Rats Aorta Reduced activity [359]
infarcted 60 min/day for 11 weeks
Male, adult (6 months) and Swim training (60 min/day, 5 days/week for
Aorta Decreased expression of gp91(phox) [387]
aged (24 months) 10 weeks)
Reduced protein expression of
Pigs Females Aortic endothelial cells Chronic exercise training for 1619 weeks [365]
p67(phox)
Young (68 months) and aged
Mice Aorta Voluntary wheel running for 1014 weeks Reduced expression and activity [360]
(2932 months)
Decreased protein expression of
Males, diabetic and young Aorta Treadmill exercise for 10 weeks [194]
gp91(phox)
17
Exercise
Anti-inflammatory actions Transient ROS generation laminar shear stress
Synapsin
AMPK
MAPKs
CREB
chronic ROS production PGC-1 AP-1 and NF-B
SOD
mitochondrial biogenesis SOD NADPH oxidase
ROS production GPX, catalase VCAM-1
TrxR1
HSPs
telomerase
proteasomes complex,
OGG1, UDG
Figure 4: Signaling pathways underlying the antioxidant actions of exercise. AMPK = AMP-activated protein kinase, AP-1 = activator protein-
1, CREB = cAMP-response-element binding, HSPs = heat shock proteins, GPX = glutathione peroxidase, MAPKs = mitogen activated protein
kinases, NF-B = nuclear factor kappa B, OGG1 = oxoguanine DNA glycosylase, PGC-1 = peroxisome proliferator-activated receptor gamma,
coactivator 1-alpha, SOD = superoxide dismutase, ROS = reactive oxygen species, TrxR1 = thioredoxin reductase 1, and UDG = uracil DNA
glycosylase.
(6) GSR. Increased activity was reported in humans [355] as the degradation of oxidatively damaged proteins [308, 391
well as in rats brain, liver, lung, muscles, heart, muscle, and 393], and therefore enhances the cellular repair processes.
testes [311, 377, 382, 383]. Other studies reported that exercise Exercise modulates the activity of DNA repair enzymes,
training produced no change in GPx activity [292, 298]. particularly oxoguanine DNA glycosylase (OGG1) and uracil
DNA glycosylase (UDG), and thus reduces the accumulation
(7) GST. Increased activity was reported in liver and salivary of nuclear 8-hydroxydeoxyguanosine (8-OHdG) and muta-
glands of mice after 8 weeks of treadmill training [136]. tions in skeletal muscles [314, 394, 395] but not brains of aged
rats [396].
(8) Thioredoxin Reductase 1. Exercise increased thioredoxin
reductase 1 (TrxR1), one of the thioredoxin system enzymes Effects of Exercise on Telomeres. Telomeres are often regarded
with direct and indirect antioxidant effects, in peripheral as the guardians of the genome. Telomere dysfunction
blood mononuclear cells in humans [205, 385]. activates p53, leading to suppression of PGC-1 and PGC-
1 promoters with consequent metabolic and organ failure
(9) NAD(P)H Oxidase. Reduced gene [386], protein expres- [397]. Ten cross-sectional and longitudinal studies described
sion [194, 360, 365, 386, 387], and activity [359, 360] have a positive association of physical activity with telomere length
been detected in humans [386] and experimental animals in immune cells and skeletal muscles [398]. The leukocyte
following endurance exercise. Also, the physically active telomere was 200 nucleotides longer in people who exercise
elderly had lower NAD(P)H oxidase activity in their vascular regularly, which roughly corresponds to a ten-year increase in
endothelial cells compared with less active subjects [133]. longevity [399]. Exercise increases the activity of telomerase
Exercise-induced adaptation of antioxidant and proox- and induces the expression of telomere repeat-binding factor
idant enzymes is highly isoform [296, 307, 370, 372, 373], 2 and Ku70 in thoracic aorta and leukocytes from mice and
tissue [311, 362, 371, 377, 378, 383], age [23, 138, 222, 297, humans [400]. However, other studies showed no association
311, 366, 384], time course [23, 230, 292, 362], and exercise or inverted U relationship of physical activity with telomere
mode specific [352, 373, 388]. Exercise modulates the three length [398] warranting further investigation. The signaling
SOD isoforms differently [296, 362, 365, 372, 373, 389] as pathways underlying the antioxidant actions of exercise are
the promoter region of SOD-2 contains more ROS-sensitive summarized in Figure 4.
binding sites [390]. Exercise-induced protein expression of Exercise training confers a myriad of physiological
SOD is time dependent; SOD-1 protein expression was benefits in aging and cardiovascular diseases through its
increased in rat skeletal muscles 48 hours after exercise, antioxidant and anti-inflammatory actions. The inflamma-
whereas SOD-2 protein content was increased after 10 and 24 tory actions of exercise are mainly exerted on adipose
hours but not 48 hours [362]. tissue (by reducing its mass and inflammatory environment),
on the immune system (by shifting immune cells towards
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http://dx.doi.org/10.1155/2016/8408479
Review Article
Cardiovascular and Hepatic Toxicity of Cocaine: Potential
Beneficial Effects of Modulators of Oxidative Stress
Manuela Graziani,1,2 Letizia Antonilli,1,2 Anna Rita Togna,1 Maria Caterina Grassi,1,2
Aldo Badiani,1,3 and Luciano Saso1
1
Department of Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, Rome, Italy
2
Drug Addiction and Clinical Pharmacology Unit, University Hospital Umberto I, Sapienza University of Rome, Rome, Italy
3
Sussex Addiction Research and Intervention Centre (SARIC), School of Psychology, University of Sussex,
Brighton BN1 9RH, UK
Correspondence should be addressed to Manuela Graziani; manuela.graziani@uniroma1.it
Received 7 August 2015; Revised 19 October 2015; Accepted 1 November 2015
Academic Editor: Jose L. Quiles
Copyright 2016 Manuela Graziani et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Oxidative stress (OS) is thought to play an important role in the pharmacological and toxic effects of various drugs of abuse. Herein
we review the literature on the mechanisms responsible for the cardiovascular and hepatic toxicity of cocaine with special focus
on OS-related mechanisms. We also review the preclinical and clinical literature concerning the putative therapeutic effects of
OS modulators (such as N-acetylcysteine, superoxide dismutase mimetics, nitroxides and nitrones, NADPH oxidase inhibitors,
xanthine oxidase inhibitors, and mitochondriotropic antioxidants) for the treatment of cocaine toxicity. We conclude that available
OS modulators do not appear to have clinical efficacy.
1. Introduction It has been suggested that OS plays an important role

in the physiopathology of various apparatuses and organs
Oxidative stress (OS) can be defined as an unbalance between including the cardiovascular system (ischemia and reperfu-
the production of reactive oxygen and nitrogen species sion injury, heart failure, atherosclerosis, hypertension, etc.)
(ROS and RNS) and the compensatory response of physio- and the liver (acute and chronic damage) [1, 2]. There is also
logical antioxidant mechanisms. The main ROS are super- evidence of significant involvement of OS in the pharmaco-
oxide (O2 ), hydrogen peroxide (H2 O2 ), hydroxyl ( OH), logical and toxic effects of drugs of abuse and particularly of
hydroperoxyl (HO2 ), peroxyl (RO2 ), alkoxyl (RO ), singlet psychostimulants such as cocaine and methamphetamine [3].
oxygen (1 O2 ), hypochlorous acid (HOCl), and ozone (O3 ) The level of OS in the aforementioned conditions can be
while the main RNS are nitric oxide ( NO), nitrogen diox- measured by a number of biomarkers, including H2 O2 , NO
ide ( NO2 ), peroxynitrite (ONOO ), nitrous acid (HNO2 ), derivatives (nitrite, nitrate, and S-nitrosothiols), isoprostanes
dinitrogen tetroxide (N2 O4 ), dinitrogen trioxide (N2 O3 ), and (deriving from the peroxidation of arachidonic acid), MDA
nitronium cation (NO2+ ). The most important sources of and other thiobarbituric acid reactive substances (TBARS),
ROS and RNS are represented by enzymatic reactions local- 4-hydroxynonenal (4-HNE), acrolein, thiol/disulfide ratio,
ized in the mitochondria, the microsomes (cytochrome P450 oxidation products of DNA (8-hydroxy-2-deoxyguanosine,
enzymes), the cytosol such as xanthine oxidase (XO), and 8-OH-G) and RNA (8-hydroxyguanosine, 8-OHD), and
the membrane-associated protein complex with its cytosolic nitrotyrosine. It is of note that, in several studies, cocaine-
subunits NADPH oxidase (Nox). The production of ROS in induced OS was evaluated by the measurement of TBARS
the phagocytes depends on the activity of peroxidases such as [49] which is considered inferior to other methods for lipid
myeloperoxidase and eosinophil peroxidase. peroxidation like the evaluation of F2-isoprostanes [10].
In the present paper, we review the literature concerning leads to vasoconstriction and increased blood pressure.
the cardiovascular and hepatic toxicity of cocaine with special Increased heart rate, heart contractility, and blood pressure
attention to the role of OS and the evidences about the may lead to an acute imbalance in oxygen supply/demand
possible modulators of OS which could have beneficial effects [37, 38]. Oxygen deficiency can be further exacerbated by
in cocaine users. the reduction in blood supply to myocardium produced
by 1-mediated constriction of coronary arteries [13]. In
2. Cardiovascular Toxicity of Cocaine turn, oxygen deficiency may lead to myocardial infarction.
Furthermore, the combination of the direct toxic effect of
The earliest case reports of cardiovascular toxicity attributed cocaine and those of norepinephrine may lead to complex
to cocaine date from the 1980s [1113]. Cocaine abuse is asso- arrhythmia [39].
ciated with both acute and chronic cardiovascular toxicity In addition to producing oxygen imbalance, cate-
[1416], including myocardial ischemia [13, 17] and infarction cholamines may damage the myocardium via at least three
[18], arrhythmias [19], and cardiomyopathy [2022]. Recent additional pathogenetic mechanisms [30, 40]. First cate-
epidemiological data indicate that cocaine is responsible for cholamines can promote cation translocation from the vas-
a sizeable proportion of emergency department visits and cular space to intracellular compartment [41] thus reducing
of sudden deaths [23, 24]. Data from 19 European countries serum K+ [42] and Mg 2+ [43]. Concurrent hypokalaemia
indicated more than 500 cocaine-related deaths in 2012 [25]. and hypomagnesaemia may lead, given the pivotal role of
Approximately 5% to 10% of emergency department visits in these cations in activity of the Na+/K+-ATPase pump [44], to
the United States have been attributed to cocaine-acute tox- further cation dyshomeostasis, which in turn may contribute
icity, chest pain being the most common symptom [15]. The to apoptosis and necrosis of cardiomyocytes. Moreover, this
upward trend in cocaine-related chest pain and myocardial mechanism [45], adding to the direct arrhythmogenic effects
infarction cases has induced the America Heart Association of cocaine described above, may facilitate the development
to draft diagnostic and therapeutic guidelines [26]. Data of arrhythmias such as atrial fibrillation and ventricular
from the relative National Cardiovascular Data Registry tachycardia.
was recently published [27]. Histopathological studies have
A second pathway responsible for cardiotoxicity is
shown that cocaine can precipitate myocardial ischemia in
related, as first hypothesized by Fleckenstein and Coworkers
the presence of coronary artery occlusion [28] as well as
in 1974 [46], to catecholamines-induced calcium overload in
of normal coronary arteries [29]. A recent review [23] of
cytosol and mitochondria of cardiomyocytes [41]. Indeed,
49 cocaine-related deaths identified coronary atherosclerosis,
stimulation of -adrenergic receptors leads to activation of
ventricular hypertrophy, cardiomegaly, myocarditis, and con-
traction band necrosis in almost a third of cases. protein kinase A (PKA) and increased Ca2+ levels in the
The pathogenesis bases of cocaine-induced cardiovascu- cytosol. This leads to phosphorylation of Ca2+ -protein sub-
lar toxicity [14, 30, 31] have been studied in detail [32, 33]. strates, including phospholamban, L-type calcium channel,
Cardiovascular cocaine toxicity can be related to its patho- ryanodine receptor, cardiac troponin I, and myosin-binding
physiological effects on the sinoatrial node, myocardium, and protein C [47, 48]. Increased cytosolic Ca2+ triggers the
vasculature, including the coronary district. release of Ca2+ in the mitochondria. The mitochondrial role
in physiological intracellular Ca2+ homeostasis as well as
2.1. Pathogenetic Mechanisms of the Cardiac Toxicity of in necrosis and apoptosis signaling is well demonstrated
Cocaine. Cocaine can damage the heart through a variety [49]. Mitochondrial Ca2+ overload impairs respiration and
of mechanisms that have been elucidated only in part. In ATP production and produces change in permeability of the
the first place, cocaine has a direct cardiotoxic effect, due its mitochondrial membrane (eventually leading to its rupture),
ability to block voltage-dependent K+ and Na++ channels in which represents critical events for the further structural
the sinoatrial node and the myocardium, leading to reduced degeneration of cardiomyocytes [50].
contractility and to prolongation of the QT interval and the Moreover Ca2+ overload (as well as ROS mitochondrial
QRS complex. It has been proposed that these two effects production, see below) is responsible for the massive opening
may produce acute myocardial ischemia and infarction also of mitochondria Permeability Transition Pores (mPTP) [51]
in absence of long-term cocaine abuse, of abnormalities in the resulting in further dysfunctional and structural degenera-
coronary arteries, and of other risk factors [34, 35]. tion of these organelles.
Cocaine can exert its toxic effect on the heart also An indirect cardiotoxic effect of catecholamines may
indirectly, through the actions of catecholamines, and in derive from action of their oxidation products, the amino-
particular of norepinephrine. Indeed, cocaine is known chromes. Notably, excessive level of circulating catechol-
to block the reuptake of catecholamines by binding the amines (and consequent saturation of the monoaminooxi-
transporters for dopamine (DAT) and norepinephrine (NET) dase and catechol-o-methyl transferase systems) may
[36]. Increased norepinephrine levels in the terminals of the cause the increased formation of adrenochrome (obtained
sympathetic nervous system lead to activation of adrenergic by oxidation of adrenaline) [40, 52] of 5,6-dihydroxy-1-
receptors. Activation of 1 adrenergic receptors located on methylindole and of adrenochrome alkaline rearrangement
the cells of the sinoatrial node and of the myocardium results product adrenolutin. In the heart the enzyme cytochrome c
in increased heart rate and contractility. Activation of 1 oxidase has been associated with adrenochrome formation
receptors located on the smooth muscle cells of blood vessels [52].
Experimental studies investigating the relationship relative to controls in perfused hearts [66]. Accordingly,
between aminochromes and cardiotoxicity [52, 53] demon- in vivo administration of mitochondria-targeted antioxidant
strated a direct toxic effect on cardiomyocytes: disturbances MitoQ had shown to prevent left ventricular (LV) diastolic
in cellular Ca2+ homeostasis and a perturbation of oxidative dysfunction (characterized by an increase in the index of LV
phosphorylation have been reported. Accordingly, increased relaxation, in LV end-diastolic pressure-volume relation, and
levels of adrenolutin were observed in death-associated in LV end-diastolic pressure) induced in rats treated with
heart failure [54]. Moreover, aminochromes are known to cocaine for 7 days [67]. Beta-AR stimulation induced both
induce redox cycling with consequent generation of ROS cAMP [68] and PKA increase both in bovine [69] and in rat
(see below). [68, 70] cardiomyocytes mitochondria.
More recently, OS and generation of ROS have been iden- Experimental data in mouse cardiomyocytes [66] have
tified as one of the most important mechanisms of cocaine- demonstrated that -AR exposure to agonist ISO leads to an
induced cardiomyocyte toxicity [6, 30, 31, 55, 56]. Increased increase in ROS, suggesting ROS production as a direct con-
expression iNOS and decreased levels of myocardial SOD sequence of activation of the cAMP-PKA signaling pathway
and catalase were found in the cardiomyocytes of patients in mitochondria. Importantly, the increase in mitochondrial
with dilated cardiomyopathy related to chronic cocaine abuse ROS production appeared to be Ca2+ -independent, since a
[20]. Indeed, ROS formation has been thought to be related selective increase of the amplitude of Ca2+ transient did not
to cocaine-induced catecholamine release [56]: 1 and increase ROS production [66].
receptors stimulation, as well as enzymatic and nonenzymatic -AR stimulation is also implicated in the impairment of
degradation of catecholamines, lead to intracellular ROS antioxidant system. Indeed, significant reduction in CuZn-
formation. SOD enzyme activity has been found in hearts from ISO-
treated rats as well as in ISO-stimulated isolated cardiomy-
2.1.1. 1 -Adrenoceptors Stimulation and Production of ROS. ocytes [71]. Moreover, a reduction in manganese-SOD (Mn-
In the plasma membranes of cardiac cells, 1 -adrenoceptors SOD) expression induced by chronic ISO was more recently
stimulation increases the activation of nicotinamide adenine found in type 5 AC (1 of 2 major AC isoforms in heart)
dinucleotide phosphate (NADPH) oxidases [57], an electron transgenic mice [72].
donor which in turn produces the superoxide anion radical
O2 formation [56, 58]. The role of NADPH-driven super- 2.1.3. OS from Catecholamines Metabolites (Aminochro-
oxide production in cocaine induced cardiac dysfunction is manes). As mentioned above, when the enzymatic catab-
well recognized [59]. Indeed, the family of Nox enzymes is olism of catecholamines is not sufficient, they can undergo
considered the major sources of ROS in the cardiovascular chemical oxidation causing additional OS [40]. The impli-
system [58, 60]: the proteins of the Nox family produce O2 cation of aminochromanes in the pathogenesis of cardiac
by transferring an electron from NADPH (or NADH) to O2 . diseases [53, 73] is also well recognized.
It is believed that Nox1, Nox2, and Nox4 are expressed signifi- Formation of superoxide anion O2 (due to an oxida-
cantly in the vascular system [61, 62] while experimental data tion pathway that involves the formation of highly reactive
in cardiomyocytes had demonstrated that the Nox subunit intermediaries o-semiquinones and o-quinones) [74] has
Nox1, but not Nox2 or Nox4, is implicated in norepinephrine- been observed in both in vivo [75] and in vitro models [74,
induced Nox-generated ROS [63]. Inhibition of Nox activity 76]. The superoxide anion O2 can cause the oxidation of
by apocynin prevented the increase in ROS production epinephrine [52, 77, 78] and of metal ions copper [79] and
and cardiac dysfunction induced by chronic administration iron [77], enhancing the oxidation of catecholamines. Thus,
of cocaine in vivo in rats [64]. Moreover, Nox activity is iron chelation can protect against the cardiotoxicity induced
associated with other ROS-inducing enzymatic sources such by the metabolites of catecholamines: an in vitro study in rat
as xanthine oxidoreductase (XOR): a fundamental role of XO ventricular cardiomyoblast assessing the potential cardiopro-
was confirmed in an in vivo experimental model of cocaine- tective effects of some chelating agents had suggested further
induced diastolic dysfunction [65], in which treatment with investigation in vivo animal models [80].
XO-inhibitor allopurinol prevented the cocaine-increase in Catecholamines metabolites can also reduce antioxidant
mitochondrial ROS levels. defences by decreasing the levels of reduced glutathione
(GSH) and increasing the oxidized glutathione (GSSG) con-
2.1.2. -Adrenergic Receptors Stimulation and Production tent as observed in adrenaline-treated isolated rat cardiomy-
of ROS. As discussed above, -adrenergic receptors (- ocytes [74, 79].
AR) stimulation activates a GTP-binding protein S, which
stimulates adenylyl cyclase (AC) to produce cAMP, which 2.2. Pathogenetic Mechanisms of Cocaine Vascular Toxicity
in turn activates PKA. It has been demonstrated that -
AR stimulation may contribute to mitochondrial ROS pro- 2.2.1. Cocaine Effect on Vascular Smooth Muscle Cells. Co-
duction in cardiomyocytes: indeed the -adrenergic ago- caine sympathomimetic activity and consequent agonist
nist isoproterenol (ISO) increased, in a concentration- and action at 1 adrenergic receptors (1 -AR) in vascular smooth
cAMP-protein kinase A-dependent manner, mitochondrial muscle cells cause contraction in the vascular system. The
O2 production in freshly isolated mouse cardiomyocytes activation of these receptors leads in fact to the formation of
and induced a twofold increase of MDA protein adducts inositol triphosphate and diacylglycerol (via phospholipase
C), which in turn cause an increase in Ca2+ entry and in 2.2.3. Cocaine Effect on Platelet Function. Whereas both
release from Ca2+ stores in smooth muscle cells [81]. Despite experimental and clinical data agree on the fact that cocaine
the different contribution of each 1 -AR subtype [82, 83] in can affect endothelial function, leading to vasoconstriction,
the regulation of contraction in different vascular districts, there is conflicting evidence about the direct effect of cocaine
the net result of 1 -AR stimulation is represented by an acute on platelets. Increased platelet activation was observed in
increase in blood pressure. vitro in rabbit platelet-rich plasma (PRP), preincubated with
cocaine hydrochloride [102] as well as in vivo in dogs treated
with intravenous cocaine [103]. The findings obtained with
2.2.2. Cocaine Effect on Endothelial Cells. A further contribu- human platelets in vitro are less consistent. At concentrations
tion in the pathogenesis of cocaine-induced vasoconstriction comparable to the systemic concentrations produced by
is due to its acute and chronic effect on endothelial cells func- lethal doses, cocaine decreased platelet aggregation induced
tion [84]. An impairment in endothelium-dependent vasore- by agonists in PRP obtained from healthy human subjects
laxation, assessed as a decrease in forearm blood flow in [104]. In contrast, two in vitro studies conducted with cocaine
response to intraarterial acetylcholine and nitroprusside, was concentrations comparable to the systemic concentrations
found in long-term users of cocaine [85]. Accordingly exper- associated with the high showed increased platelet activa-
imental studies [86, 87] demonstrated a cocaine-induced tion in whole blood preparations [105, 106], whereas other
endothelial dysfunction with a decrease in NO release and studies found no effect of cocaine [107]. Finally, increased
in the constitutive enzyme NO-synthase (eNOS) content, as platelet expression of surface P-selectin was found in blood
well as an increase in endothelin-1 (ET-1) production and ET- samples from chronic cocaine users [107]. It is possible
1 receptor type-A (ETA R) protein expression [84]. Increased that these discrepancies were due to differences in substrate
number of circulating endothelial cells (CECs) indicat- (animal versus human platelets), in model (in vitro versus in
ing endothelial dysfunction was recently demonstrated in vivo), in methodology (whole blood versus PRP), and in the
cocaine abusers [88]. Furthermore, enhancement of cocaine- concentrations of cocaine used.
induced vasoconstriction was observed after N(G)-nitro-L- In vivo studies in healthy subjects [108] and in chronic
arginine methyl ester-induced inhibition of NO synthesis in cocaine user [109], although not univocally [110], gave evi-
vitro [89]. dence of activation of platelets, assessed by increase of P-
Concomitant decrease in NO-induced vasodilatation selectin expression [109] and of soluble CD40L, a transmem-
and ET-1-induced vasoconstriction may enhance 1 -AR- brane molecule mainly expressed by activated platelets [111].
mediated vasoconstriction. It has been suggested that inhibi- Due to physiological interaction of platelets with vascular
tion of eNOS expression may partially derive from high levels endothelium and circulating blood cells such leukocytes, it
of ET-1 through a PKC-mediated pathway in endothelial may be argued that an indirect, rather than a direct, mecha-
cells [90] and/or through ET-1 receptor type-A (ETA R) nism of action is involved in the cocaine platelets activation.
stimulation, which in turn can increase ROS production [91, Indeed, the above mentioned cocaine-induced vasospasm,
92]. consequent increase in shear stress, and endothelial dys-
Chronic cocaine exposure and consequent endothelial function may induce a platelet activation, as demonstrated
dysfunction may precipitate early atherosclerosis [93] and by release of constituents of their -granules [106] and of
the persistence of endothelial cell damage beyond its acute thromboxane A2 [99]. A further contribution may derive
effect on the blood vessels can further increase cardiovascular from VWF interaction with the platelet receptor GPIb and
risk in cocaine abusers [88, 94]. Since that ET-1 increase their subsequent activation [108].
was significantly associated with atherosclerotic lesion [95], it
may be argued that it plays a fundamental role in the cocaine-
2.2.4. OS and Cocaine-Induced Endothelial Toxicity. Besides
induced vasoconstriction at sites of significant stenosis [96].
cardiomyocytes, also endothelial cells (ECs) may release ROS,
Although discrepant results were also reported [97],
mainly due to its presence of enzymes such as XO, NOS,
probably due to methodological differences, there is some
mitochondrial MAO, and NAPDH oxidase. Some data in
evidence indicating that cocaine may exert an inhibitory
literature indicate a direct or an indirect cocaine action
effect on the production of prostacyclin (PGI2 ) by endothelial
on endothelial cells, both on enzymes expression and on
cells [98, 99]. Since the release of both vasodilatatory PGI2
mitochondrial function.
and NO may result from stimulation of ET-1 receptor type-
B1 (ETB1 ) receptors on endothelial cells, it is possible that
the inhibitory effect of cocaine on NO release also extends 2.2.5. XO and Production of ROS. In bovine aortic endothelial
to PGI2 release [100]. cells, it has been observed that shear stress induced an
Another important factor that can contribute to cocaine enhancement of xanthine-dependent O2 production [112],
prothrombotic action is its direct action on endothelial cells associated with a decrease in xanthine dehydrogenase (XDH)
secretion of von Willebrand factor (VWF). Indeed, a recent in protein. Furthermore, inhibition of the Nox decreased XO
vitro study [101] demonstrated that cocaine and its metabo- levels and prevented the increase of O2 , highlighting the
lites induced VWF secretion in a concentration-dependent central role of Nox in modulating endothelial production
manner from three endothelial cell types (human umbilical of ROS. It may be suggested that vasoconstriction effect
vein, brain microvasculature coronary artery endothelial of cocaine and consequent blood shear stress may trigger
cells). endothelial ROS production via Nox/XO enzyme.
In agreement with this hypothesis, cocaine-induced car- 2.2.9. ROS Production by Mitochondrial Nox4. Another pos-
diac dysfunction (alteration of cardiac output and stroke sible source of ROS in endothelial mitochondria is Nox4
volume) was found to be associated with increased Nox [124], generating a higher hydrogen peroxide to superoxide
and XOR activity in vivo study in rats [64]. Furthermore, ratio than Nox1 and Nox2. Nox4 involvement in endothelial
apocynin or allopurinol treatment inhibited the cocaine- cells process and in responses to hypoxia and OS is well
induced cardiac alteration and the myocardial production recognized [125]; moreover it has been demonstrated that
of O2 confirming the role that Nox-derived ROS play in the expression or activity of Nox4 is increased in response
modulating ROS production by XO [64]. to the proinflammatory mediators TNF- [126]. While a
cocaine-induced increase in Nox has been demonstrated in
2.2.6. Nitric Oxide Synthase and Production of ROS. As cardiac tissue [64], to date no data in literature are present
mentioned above, cocaine-induced decrease in endothelial on the effects of cocaine on Nox at endothelial level. An
NO release and in the constitutive enzyme eNOS content indirect effect of cocaine activation of Nox may derive from
has been observed [84]. A contribution to this cocaine the cocaine induction of the TNF- expression, observed in
endothelial toxic effect may derive from its increasing action bovine aortic endothelial cells [127]. Moreover a Nox increase
in Nox, which in turn (besides the increase in O2 ) may and the consequent endothelial superoxide production were
induce oxidation of tetrahydrobiopterin, a cofactor of NO also found in bovine aortic endothelial cells exposed to
synthase: as a consequence eNOS uncoupling leads to the oscillatory shear stress [112].
observed reduction in NO synthesis and to an enhancement
in O2 [113]. 3. Cocaine Hepatotoxicity
Cocaine abuse is known to induce acute [128132] and
2.2.7. ROS in Endothelial Mitochondria. Besides the well- chronic [130, 131, 133] liver toxicity. Clinical manifestations
recognized energy-producing activity, notably mitochon- of cocaine-induced hepatic damage range from elevation of
dria are the major source of cellular reactive oxygen [114] liver enzyme levels in chronic users [131, 133, 134] to acute liver
both in cardiomyocytes and in EC [40, 115]. An impor- failure associated with hepatitis [128], to fulminant liver fail-
tant role of endothelial mitochondria in pathogenesis of ure associated with acute rhabdomyolysis [132] or thrombotic
endothelial dysfunction is due to their role in signaling microangiopathy [128]. Histopathological examination has
cellular responses, among which the production of ROS shown midzonal [132] and periportal [130] necrosis, as well as
[116]. steatosis in the surviving hepatocytes [131]. The involvement
of OS in cocaine liver toxicity has been reviewed recently
2.2.8. Mitochondrial Monoaminooxidase and Production [135, 136].
of ROS. Among the sources of mitochondrial ROS, the In vivo animal studies suggest the involvement of cocaine
monoamine oxidase (MAO) family, located to the outer mito- metabolites in the genesis of hepatotoxicity. Experiments
chondrial membrane, causing the oxidative deamination of conducted in rats [137] and mice [138, 139] have shown
catecholamines, results in hydrogen peroxide (H2 O2 ) forma- that cocaine-induced hepatotoxicity is at least partly depen-
tion [117]. Accordingly, experimental data in literature indi- dent on cocaine N-demethylated metabolites norcocaine
cate an increase in H2 O2 cardiac production after the sympa- (NCOC), and N-hydroxynorcocaine (N-OH-NCOC) and
thomimetic drug amphetamine administration [118]. In this norcocaine nitroxide (NCOC-NO ) [139, 140]. Inhibition of
regard a recent in vitro study has demonstrated in human cytochrome P-450 (CYP450) mediated activation of cocaine
pulmonary EC [119] exposed to cocaine a significant increase metabolism produced a significant inhibition of hepatotoxic-
in H2 O2 production; due to the modulating action of H2 O2 ity in mice in vivo [138] and ex vivo in liver microsomes [141],
on endothelium functions such as endothelium-dependent while in vivo induction of CYP450 activity enhanced the
vasorelaxation, apoptosis, and remodeling [120] it may be cocaine hepatotoxicity [142]. Studies in mice demonstrated
argued that cocaine-induced increase in catecholamines and that cocaine reduces GSH levels in the liver [143, 144] and that
in the consequent MAO-mediated H2 O2 production in depletion of intracellular GSH concentrations exacerbated
endothelial mitochondria could further enhance endothelial cocaine hepatotoxicity [144]. Finally, it has been shown
dysfunction, contributing to cocaine toxic effects on vascular that pretreatment with the GSH precursor NAC exerted a
district. protective effect against cocaine hepatotoxicity in mice [145],
An important contribution to OS may derive from the while pretreatment with endotoxin lipopolysaccharide (LPS)
process, namely, ROS-induced ROS release (RIRR) [120]: this led in Kuppfer cells to the formation of NO which exacerbated
process makes a significant amplification to ROS production cocaine toxicity [146].
[121]. Briefly, OS in the mitochondria may trigger the opening Given that the synthesis of both NCOC-NO and GSH
of mitochondrial transition pore that leads to a further requires the presence of the cofactor NADPH, it has been
increase in ROS generation. RIRR phenomena have been hypothesized that a decrease in hepatocytes NADPH content
recognized in both physiological (promoting an elevation in and consequent impairment of antioxidant system may con-
the cell tolerance to OS, until the destruction of impaired- tribute to enhance OS [147].
function mitochondria) [122] and pathological conditions The ability of cocaine to deplete intracellular GSH may
such as cardiac ischemia-reperfusion [123] associated with also depend on the effects of its N-oxidative metabolites on
acute myocardial infarction. the mitochondrial function of the hepatocytes. In vivo studies
in rats [148, 149] have shown that these metabolites can considered a promising drug for these pathological condi-
decrease GSH levels and membrane potential and increase tions [158, 160]. A recent double-blind placebo-controlled
ROS production in the mitochondria [9]. Mitochondrial trial failed to demonstrate that NAC reduces cocaine use in
generation of ROS has been shown also in isolated mouse cocaine-dependent subjects but showed some evidence that it
liver mitochondria treated with NCOC, N-OH-NCOC, and can prevent relapse in individuals who had already achieved
NCOC-NO but not with cocaine [150], confirming the abstinence from cocaine [161]. Thus NAC may be useful as
fundamental role of the N-oxidative metabolites of cocaine a relapse prevention agent in abstinent cocaine-dependent
in OS-mediated hepatotoxicity. subjects [161].
Ex vivo and in vitro studies have confirmed the in vivo The mechanism of action of NAC in the aforementioned
data. Cocaine-induced GSH depletion and GSSG production conditions is complex and not completely elucidated. The
were observed in cultures of mouse and, to a lesser extent, main activities contributing to its efficacy seem to be related
in rat cocaine-treated hepatocytes [151, 152] with mouse to (i) its capability of providing cysteine which is the lim-
being more sensitive to cocaine hepatotoxicity, as observed iting amino acid in GSH production; (ii) the exchange of
in cultured liver slices from different species [153]. In primary extracellular cysteine provided by NAC with intracellular
cultures of rat hepatocytes treated with the CYP-450 inducer glutamate by the cysteine/glutamate transporter causing the
phenobarbital, it has been observed that cocaine-induced activation of presynaptic mGluR2/3 receptors which inhibit
alteration in the thiol redox equilibrium may be crucial for glutamatergic neurotransmission and excitotoxicity [162,
the development of hepatocyte toxicity and consequent LDH 163]; (iii) direct antioxidant activity related to its capability
release [154]. Moreover cocaine-treated hepatocytes had been of scavenging radicals such as hydroxyl and peroxynitrite
associated with decrease in catalase and Mn-SOD [155]. [164]; (iv) induction of the expression of antioxidant enzymes
Further confirmation of implication of OS is derived from rat through the nuclear E2-related factor (Nrf2)/ARE system
hepatocyte model of cocaine cytotoxicity in which a partial [165]. It is of note that the Nrf2 pathway represents a promis-
prevention of cytotoxicity by NAC [156] and by deferoxamine ing therapeutic approach to restore the redox balance in the
(a ferric iron chelator) was observed [152, 156]. CNS and in other organs and that only a few Nrf2-activating
In summary, human and experimental data provide compounds have been tested in a clinical setting until now
evidence of direct toxic effects of cocaine on the hepatocytes. [166].
This hepatotoxicity appears to be dependent mainly on N-
oxidative metabolites of cocaine. Impairment of the antiox-
4.2. SOD Mimetics. SOD is a very important antioxidant
idant system as depletion of intracellular and mitochondrial
enzyme in mammals existing in three different physiological
GSH also contributes to cocaine hepatotoxicity.
forms: MnSOD in the mitochondrial matrix, CuZnSOD in
the cytoplasm, and CuZnSOD in extracellular fluids [167].
4. Potential Therapeutic Effects of OS Given the central role of the superoxide radical in oxidative
Modulators in Cocaine Abuse pathological phenomena, different drugs with SOD activity
were developed [168].
Antioxidants or modulators of OS as they are often referred An infusible form of human manganese SOD (rMn-SOD)
to due to their possible prooxidant activity can be classified in as new therapeutic option capable of crossing cell membranes
low (LMW) and high (HMW) molecular weight and further was recently proposed for dilated cardiomyopathy caused by
subdivided into endogenous or exogenous. The exogenous cocaine [20].
ones can be natural or synthetic [2]. Several evidences
indicate that some of these compounds could have beneficial
4.3. Nitroxides and Nitrones. Most antioxidants acting as spin
effects on cocaine-induced toxic effects.
traps have a nitroxide or nitrone nucleus.
Nitroxides are nonmetal catalytic antioxidants. One of
4.1. NAC. NAC has been used for many years in the treat- the most common is Tempol (4-hydroxy-2,2,6,6-tetram-
ment of acute paracetamol intoxication, as a mucolytic for ethylpiperidine-N-oxyl) with SOD and catalase enzymatic
chronic obstructive pulmonary disease and as a protec- activities which can be administered orally and can cross
tant in contrast-induced nephropathy. NAC can scavenge biological membranes and react with ROS intracellularly and
directly ROS and particularly the hydroxyl radical OH and within mitochondria [169].
hypochlorous acid but due to its high first pass metabolism Nitrones are potent antioxidants capable of forming
and low bioavailability acts mainly as a precursor of GSH stable nitroxyl radicals when reacting with oxygen radi-
in many organs, including the liver [145] and the brain cals. Several nitrones, including -phenyl-tert-butylnitrone
[157]. As mentioned before, mice pretreatment with NAC had (PBN), have been shown to have neuroprotective properties
a protective effect against cocaine-mediated hepatotoxicity in experimental animal models of stroke, Parkinsons and
[146]. Importantly, NAC treatments appear to be safe and Alzheimers disease [170], as well as in preclinical models
tolerable both in preclinical and in clinical studies [158, 159]. of CNS injury [171]. Treatment with Tempol can attenuate
GSH is a very important endogenous antioxidant in the OS in the prefrontal cortex and in the nucleus accumbens
brain and it has been known for a long time that its level [172] and inhibited cocaine self-administration in vivo in rats,
decreases in neuropsychiatric disorders such as depression, suggesting that reinforcing effects of cocaine are mediated, at
schizophrenia, and bipolar disorder [160] and is currently least in part, by ROS [173].
To the best of our knowledge, no data are present in production, membrane permeabilization with release of
literature on efficacy of nitroxides and nitrones on cocaine- cytochrome C, and subsequent cell death: MitoQ can prevent
induced cardiac or hepatic injury. these abnormalities protecting against cardiac dysfunction
[67].
4.4. Nox Inhibitors. As mentioned above, Nox are among Preparation of small cell-permeable peptides (SS-
the best characterized sources of superoxide and the pre- peptides) which accumulate in the mitochondria and bind to
dominant ROS producing enzymes in the vascular smooth the inner membrane thanks to the presence of basic amino
muscle cells, in the myocardium, and in blood vessels [174]. acids positively charged at physiological pH had shown to
Hypertension, atherosclerosis, hyperlipidemia, and diabetes protect mitochondria against oxidative damage [185, 186].
are associated with endothelial dysfunction probably medi- The use of these peptides to deliver antioxidant molecules
ated by Nox driven production of ROS [175]. Compared to into mitochondria and provide protection against cocaine
radical scavengers, Nox inhibitors could be more effective induced OS has been proposed [187].
because they are able to block the formation of ROS at the
source [176]. 4.7. Deferoxamine. NAC and deferoxamine (DFO, an iron
Several Nox inhibitors are currently available, includ- chelating agent that prevents ROS generation by inhibit-
ing apocynin, which is an orally active natural compound ing the Fenton reaction) protected rat hepatocytes in cul-
obtained from the roots of Picrorhiza kurroa capable of ture against the OS induced by cocaine, confirming the
inhibiting Nox activation probably by preventing the assem- involvement of oxygen radicals in cocaine-induced necro-
bly of Nox2 with p47phox [177, 178]. sis/apoptosis [188]: hepatocytes were preincubated for 24 h
Importantly it was reported that apocynin can prevent either in the presence or in the absence of NAC or DFO
cocaine-induced myocardial damage in rats [64] providing a and exposed for another 24 h to increasing concentrations of
good evidence for the therapeutic potential of Nox inhibitors cocaine together with NAC or DFO. Pretreatment with defer-
in the treatment of cocaine cardiotoxicity [179]. oxamine complex with iron ion (III), aminoguanidine, and
N-methyl-d-glucamine dithiocarbamate complex with iron
4.5. XO Inhibitors. Allopurinol is a well-known inhibitor of ion (II) produced a marked inhibition of the hepatotoxicity
XO used for decades for the treatment of gout [180]. It can induced by a single administration of cocaine, as indicated
prevent OS by inhibiting XO and also by scavenging directly by histopathological examination, alanine aminotransferase
hydroxyl free radicals. activity, and nitrite/nitrate levels [183].
Clinical studies showed that allopurinol has beneficial
effects in chronic kidney disease patients [181]. XO inhibition 4.8. Selenium. In rats, pretreatment for 4 weeks with sele-
by allopurinol (or the active metabolite oxypurinol) could nium reversed both the OS and the contractile dysfunction
be beneficial in hyperuricemic patients with congestive heart induced by repeated (7 days) cocaine administration in rats
failure [182]. Unfortunately, allopurinol is not devoid of side [189]: cardiac function was evaluated by cardiac index and
effects such as severe skin reactions and renal impairment left ventricular fractional shortening measured by echocar-
[182]. diography.
Allopurinol and 1,3-dimethylthiourea, a scavenger of
hydroxyl radical, produced a potent inhibition of hepatotox-
4.9. Minocycline. Minocycline, a second generation tetracy-
icity induced by cocaine in adult male mice [183].
cline, has been found in rats to prevent cardiac myocyte
Apocynin or allopurinol prevented cocaine-induced car-
death (associated with an increase in OS evidenced by
diac alteration by restoration of cardiac output, stroke vol-
low GSH/GSSG ratio and increased levels of 4-hydroxy-2-
ume, and fractional shortening, associated with a reduction
nonenal) induced by prenatal cocaine exposure [190].
of the myocardial production of superoxide anions and an
enhancement of catalase activity [55] suggesting that NADPH
and XO can act synergically to form myocardial ROS and that 4.10. Auranofin. Pretreatment of mice with auranofin (a well-
their inhibition could prevent the onset and progression of known disease-modifying gold compound used for rheuma-
cocaine-induced left ventricular dysfunction [55]. toid arthritis recently proposed also for other therapeutic
applications) [191] showed an interesting protective effect
4.6. Mitochondriotropic Antioxidants. Cationic triphenyl- against hepatic injury caused by cocaine by inducing over-
phosphonium (TPP) derivatives such as MitoQ have high expression of heme oxygenase-1 (HO-1), an important OS
affinity for the inner mitochondrial membrane that is neg- marker responsible for heme degradation [192]. Unfortu-
atively charged. They can cross the blood-brain barrier even nately, auranofin is not devoid of toxic effects [193] but this
if the uptake in the brain appears less than for other organs result indicates that it could be worthwhile to search for less
[184]; TPP compounds can be administered orally and did toxic inhibitors of HO-1 to reduce hepatic injury induced by
not raise safety concerns so far [184]. MitoQ was protective cocaine.
in a large number of cell models of mitochondrial OS and
has been shown to be well tolerated, orally active, and safe 4.11. Amiodarone. Amiodarone is a class III antiarrhythmic
in humans [185]. drug used in heart failure and postischemic heart capable
Cocaine can be toxic for mitochondria, causing formation of protecting cardiac myocytes against OS [194]. However,
of ROS, impaired electron transfer, suppression of ATP amiodarone pretreatment did not affect the seizure incidence
or mortality in mice treated with high doses of cocaine [195]. markers may not be appropriate for specific diseases
The efficacy of amiodarone in the treatment of cocaine- in which OS involves specific tissues or organs or
induced dysrhythmias is still undefined [196] and further organelles.
studies should be performed on this and other antiarrhyth- (6) Some antioxidants can act as prooxidants in certain
mic drugs. conditions or have adverse side effects.
4.12. Trolox. Trolox, a hydrophilic analog of vitamin E with In conclusion the effect of modulators of OS on the
well-known antioxidant properties [197], was found to reduce activity and toxicity of cocaine should be further studied in
the production of ROS induced by exposure to cocaine and order to develop new drugs useful for the treatment of OS-
the neurotoxic effect of the HIV-1 transactivating protein Tat mediated cocaine toxic effects.
[198].
Abbreviations
5. Conclusion
CNS: Central nervous system
In the present paper, several studies were reported demon- GSH: Glutathione
strating the important role of OS in the activity and toxicity HO: Heme oxygenase
of cocaine with special attention to its cardiovascular and MDA: Malondialdehyde
hepatic effects. Several evidences were reported showing the NAC: N-Acetylcysteine
potential beneficial effects of antioxidants or modulators Nox: NADPH oxidase
of OS (as they are referred to considering their possible NO: Nitric oxide
prooxidant effects in certain conditions) even if it should be OS: Oxidative stress
kept in mind that, like in the case of NAC, their mechanism RNS: Reactive nitrogen species
of action, which is often complex and not always fully ROS: Reactive oxygen species
elucidated, is not only related to their antioxidant activity. SOD: Superoxide dismutase
Unfortunately, for most of these modulators of OS the XO: Xanthine oxidase.
preclinical and clinical studies are limited and do not allow
drawing definitive conclusions on their efficacy in relation to
cocaine toxicity. Even in the case of NAC limited clinical trials
Conflict of Interests
were performed in cocaine users and the biomarkers of OS The authors declare that there is no conflict of interests
were not considered with sufficient attention. regarding the publication of this paper.
Other studies should then be performed also considering
the difficulty to demonstrate the clinical efficacy of modu-
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http://dx.doi.org/10.1155/2016/3578368
Review Article
Oxidative Stress in Shiga Toxin Production by
Enterohemorrhagic Escherichia coli
Katarzyna Licznerska,1 Bohena Nejman-FaleNczyk,1 Sylwia Bloch,1 Aleksandra Dydecka,1

Gracja Topka,1 Tomasz Gdsior,1 Alicja Wwgrzyn,2 and Grzegorz Wwgrzyn1
1
Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
2
Laboratory of Molecular Biology (Affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdansk, Poland
Correspondence should be addressed to Grzegorz Wgrzyn; grzegorz.wegrzyn@biol.ug.edu.pl
Received 29 July 2015; Accepted 30 September 2015
Copyright 2016 Katarzyna Licznerska et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Virulence of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins. These toxins are encoded
in genomes of lambdoid bacteriophages (Shiga toxin-converting phages), present in EHEC cells as prophages. The genes coding for
Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production.
Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce
lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other
factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with
H2 O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C
treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural
conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2 O2 excretion by either neutrophils in
infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production
is described in this paper, and the bacterial altruism and Trojan Horse hypotheses, which are connected to the oxidative stress,
are discussed.
1. Introduction: Enterohemorrhagic most severe of them, the hemolytic-uremic syndrome (HUS)

Escherichia coli Strains and Shiga [2]. The most common symptoms of this syndrome are acute
Toxin-Converting Phages renal failure, anemia, and thrombocytopenia; however, other
organs such as lung, pancreas, and heart may also be affected
Escherichia coli is a bacterial species commonly known as a [4]. Furthermore, some patients suffer from the disorders of
commensal occurring in the mammalian intestine [1]. This is the central nervous system [4].
true in most cases; however, some E. coli strains are capable The main virulence factors causing EHEC-mediated HUS
of causing disease in humans. One example of pathogenic E. are Shiga toxins, produced by the infecting bacteria. These
coli is a series of strains called Shiga toxin-producing E. coli toxins are hexameric proteins, composed of a single A-
(STEC) [2, 3]. subunit and five identical B subunits [5]. The main receptor,
Among STEC strains (defined as E. coli producing Shiga called Gb3 and occurring on the surface of many types of
toxins), the most dangerous for humans is the subset classi- eukaryotic cells, is recognized by the B-subunits. The toxin
fied as enterohemorrhagic Escherichia coli (EHEC, defined as enters cells by endocytosis, which is followed by its retrograde
E. coli causing bloody diarrhea) [2, 3]. Infection of humans by transport from the early endosome through the Golgi-
EHEC strains causes hemorrhagic colitis (HC) and in some apparatus and to the endoplasmic reticulum. The specific
patients it may result in various complications, including the proteolytic cleavage of the A-subunit results in the release of
the A1 polypeptide from the A2 fragment attached to the B SOS regulon (Figure 1(b)). However, the phage cI repressor
pentamer. A1 is the actual toxin that is translocated from the resembles LexA (Figure 2) and it is also degraded under the
ER to the cytoplasm [6, 7]. The Shiga toxin A1 polypeptide SOS stress response, causing derepression of bacteriophage
is an N-glycosidase that depurinates a single adenine residue promoters, excision of the prophage, and subsequent lytic
(A4324) within the -sarcin/ricin loop of the 28S rRNA development of the virus. Importantly, in the case of Stx
[8, 9]. This modification results in an inhibition of amino- phages, expression of the stx genes proceeds together with
acyl-tRNA binding to the ribosome and cessation of protein other phage genes [17, 20, 21] (Figure 1(a)). It is worth
synthesis, which leads to cell death [5]. mentioning that RecA-independent induction of Shiga toxin-
Since cattle are resistant to Shiga toxins, due to the lack converting prophages by chelating agents, like EDTA, has also
of the Gb3 receptor, they serve as a natural reservoir of STEC been reported [22]. In conclusion, production of Shiga toxins
strains. However, any cattle-derived products contaminated requires induction of Stx prophages, caused by either any
by STEC, and particularly EHEC, may cause severe human stress conditions provoking the SOS response or by chelating
infections, occurring usually as outbreaks. A few years ago agents.
(in 2011) such an outbreak took place in Germany, where over Under laboratory conditions, induction of lambdoid
4,000 patients developed severe symptoms and 54 of them prophages is relatively easy, and standard methods for the
died [1015]. Contaminated fenugreek and lentil sprouts were efficient induction include UV-irradiation and treatment
recognized as the source of the infection [13], indicating with antibiotics that interfere with bacterial DNA replication,
that unwashed or improperly washed vegetables, especially like mitomycin C [20, 21]. Such treatments lead to prophage
those coming from the so-called ecological farming where excision in a large fraction, if not most, of lysogenic cells
only natural fertilizers (including those coming from cattle) in a bacterial population. Nevertheless, when infection of
are used, may be a significant source of such outbreaks. humans by EHEC is analyzed, one should consider prophage
In that case, the strain of the O104:H4 serotype, which induction conditions which can naturally occur in human
caused the outbreak, had a combination of virulence factors intestine. Obviously UV-irradiation is very unlikely there,
characteristic of enteroaggregative E. coli (EAEC) and EHEC and high concentrations of antibiotics may be administered
[12, 14]. The high virulence of this particular strain could only to patients subjected to intensive therapy, while symp-
be ascribed to enhanced adhesion, survival adjustment, toms of EHEC infection appear also in nontreated persons.
antibiotic resistance, and Shiga toxin production [12]. Moreover, other inducers of prophage excision, like EDTA
Interestingly, genes coding for Shiga toxins (stx genes) are [22], irradiation with 60 Co [27], or high hydrostatic pressure
located in genomes of prophages rather than in actual bac- [28], are also unlikely to occur in the human gut. Therefore,
terial genome [16, 17]. Bacteriophages bearing the stx genes, an important question arose: what are factors or agents
called Shiga toxin-converting phages or Stx phages, can that can induce Shiga toxin-converting prophages in EHEC-
lysogenize E. coli strains making them STEC. All Stx phages infected human intestine? Understanding the mechanism
described to date belong to the family of lambdoid phages, of stimulation of Shiga toxin production might lead to
viruses having genomes organized in a manner similar to development of novel methods for prevention or treatment
that found in bacteriophage [17]. The genome of a lamb- of EHEC-caused diseases, as well as deciphering a biological
doid phage consists of blocks of genes coding for proteins
role for maintaining the Stx prophages in bacterial genomes.
responsible for specific functions. This makes recombination
and exchange of genes between various phages relatively easy
and leads to mosaicism of genomes of lambdoid phages [18]. 2. Hydrogen Peroxide as an Inducer of
In genomes of Shiga toxin-converting phages, the stx genes Shiga Toxin-Converting Prophages
are present between the antiterminator gene and the genes
coding for proteins causing cell lysis (Figure 1(a)). There were various attempts to find conditions which both
As long as the Stx bacteriophage is present in the E. induce Stx prophages and are likely to occur in the human gut.
coli host as a prophage, vast majority of its genes, including Different conditions, factors, and agents (including high and
stx genes, are silent due to the repression caused by the low temperatures, high salt concentrations, chelators, 60 Co,
phage-encoded cI protein [1921]. Under such conditions, high hydrostatic pressure, nitric oxide, and starvation) were
Shiga toxin is not produced. Effective expression of stx genes, tested, and the results of these studies have been summarized
together with all genes required for lytic development of [17]. Most of the tested conditions either did not induce
the bacteriophage, occurs only after prophage induction, lambdoid prophages or were unlikely to occur in human
though Shiga toxin 1 may also be produced under conditions intestine.
of low iron levels due to the presence of the Fe-sensitive Under conditions of bacterial infection, including infec-
promoter upstream of the stx1 locus [17, 20]. The prophage tion of the human gut, neutrophils are the first cells of the
induction occurs generally due to activation of the bacterial immune system which attack the pathogens. Among other
SOS response which is a defensive mechanism provoked bactericidal mediators, neutrophils excrete hydrogen perox-
by any conditions causing appearance of single-stranded ide to weaken bacterial cells. This oxidative stress-inducing
DNA fragments. The RecA protein recognizes such fragments agent is dangerous for bacteria that are much more sensitive
and is activated to stimulate the self-cleavage of the LexA to it than eukaryotic cells. However, it was demonstrated that
repressor (bearing the peptidase domain in its structure), such an action of neutrophils enhances production of Shiga
which under normal conditions inhibits expression of the toxins by EHEC strains [29]. Subsequent studies indicated
int Recombination N cI Replication Q Shiga toxins Lysis Morphogenesis
cIII N cI cro cII O P Q stxA stxB S R Rz Rz1

oR 3
oR 2
oR 1
tL1 tM tR 1 tR 2 tR
pL pM pR pR
+ + +
Lysogeny Lytic development Shiga toxins
cI repressor cI cleavage
RecA
Activation
ssDNA
RecA
DNA damage
(a)
LexA repressor LexA cleavage
SOS genes SOS genes

RecA
Activation
ssDNA
RecA
DNA damage
(b)
Figure 1: Schematic map of a Shiga toxin-converting phage genome. At the top of (a), regions bearing genes for particular phage functions
are shown (as they appear in a prophage). The region containing genes involved in regulation of phage development, DNA replication, Shiga
toxin production, and cell lysis is enlarged and shown in more detail. Major transcripts are shown by arrows, with arrowheads demonstrating
directionality of transcription, and promoters marked by short vertical lines at the beginning of transcripts. Terminators are marked by vertical
lines crossing the transcript lines. The cI repressor binds to R1 , R2 , and R3 operator sites, repressing L and R promoters and stimulating
its own promoter M . When DNA is damaged, single stranded DNA (ssDNA) fragments appear which are recognized by RecA protein. This
activates RecA to switch to the RecA form, able to stimulate self-cleavage by the cI repressor. Inactivated cI can no longer repress L and
R and M is not activated. This leads to effective transcription from L and R , prophage excision, and expression of vast majority of phage
genes, including those coding for Shiga toxin. (b) represents a similar mechanism leading expression of the SOS regulon which under normal
growth conditions is repressed by the LexA protein. Phage cI repressor resembles LexA; thus under conditions of the SOS response, induction
of the prophage occurs.
that hydrogen peroxide, when added to cultures of bacteria gene located in the place of the natural locus [30]. Very
lysogenic for various Shiga toxin-converting phages, is a similar results were obtained when natural isolate of EHEC
potent inducer of the prophages [30]. This was true for was tested instead of laboratory strains. Again, hydrogen
bacteriophage as well as for different Stx phages. Moreover, peroxide-mediated induction of the Stx prophage and effi-
the prophage induction was accompanied by synthesis of cient production of Shiga toxin were observed [31]. Therefore,
considerable amounts of the fusion protein, encoded by a the oxidative stress, mediated by hydrogen peroxide, leads to
Cleavage site
LexA (E. coli MG1655) S119 K 156
A84/G85
1 65 114 180 202
LexA DNA-binding Peptidase S24-like
Cleavage site S150 K 193

cI ()
1 24 77 A112/G113 145 218 237
HTH 3 Peptidase S24-like
Cleavage site S141 K 178

cI (933W)
1 33 73 L107/G108 136 203 235
HTH 3 Peptidase S24-like
Figure 2: Comparison of domain structures of E. coli LexA protein and cI repressors of bacteriophage [19] and Shiga toxin-converting
bacteriophage 933W [23, 24]. Two domains of these proteins are shown, and crucial amino acid residues are marked. Upon stimulation by
the activated form of RecA (RecA ) both LexA and cI cleave their own molecules (at indicated positions) by the peptidase S24-like domains.
The models were prepared using the DOG 1.0: Illustrator of Protein Domain Structures software [25].
pR promoter
TTGACT GATAAT A
OxyR 35 10 +1
cI -ctcatacgttaaatcTA T C A C C G C A A G G G A T A a a t a t c T A A C A C C G T G C G T G T T GactatttTACCTCTGGCGGTGATAatggttgca- cro

o R3 o R2 o R1
+1 10 35
A TTAGAT ATAGAT
pM promoter
pR promoter
TTGCAA TAATAC A
OxyR? 35 10 +1
933W cI -cT G A A C C A TG A G T A C GatactaaagcacttgcaA A A A C T T T C A G T T C AaccataataC G T A C T G A A A G T A C Gaaaaaggatattcct- cro

o R3 o R2 o R1
+1 10 35
A TATGAT AAAGTC
pM promoter
Figure 3: The R -M regions of phage and Shiga toxin-converting phage 933W. Structural elements of each promoter are indicated, and
R1 , R2 , and R3 operator sequences are marked. The OxyR binding to the R -M region (demonstrated experimentally [26]) is shown as a
solid oval, and a putative (not verified experimentally) OxyR binding to the 933W R -M region is suggested by a dashed oval.
stimulation of expression of genes. Since such conditions 31]. Moreover, while prophage induction by UV-irradiation
can occur in human intestine, the oxidative stress is a likely or mitomycin C caused a lysis of bacterial cultures in a
candidate for a natural inducer of Shiga toxin-converting few hours, no such phenomenon could be detected after
prophages. treatment with hydrogen peroxide [30]. Subsequent calcu-
An interesting observation in studies on both laboratory lations of the efficiency of prophage induction have shown
Stx lysogens and natural isolates of EHEC was that the that while low concentration (1 g/mL) of mitomycin C
maximal efficiency of prophage induction occurred at a final caused initiation of the lytic phage development in about
H2 O2 concentration of 3 mM, and further increases in H2 O2 1030% of cells (depending on the kind of the Stx phage),
concentrations caused a decrease in induction efficiency [30, the value of this parameter was as low as 0.031.6% at the
Inactive OxyR Active OxyR
OxyR
cI cI cI cI
OxyR
cI cro cI cro
oR3
oR2
oR1
oR3
oR2
oR1
+ +
pM pR pM pR
OxyR
cI cro cI cro
oR3
oR2
oR1
oR3
oR2
oR1
+
pM pR pM pR
(a) Normal growth conditions (b) Oxidative stress conditions
Figure 4: A model for OxyR-mediated modulation of lambdoid prophage maintenance under normal growth conditions and oxidative stress.
Under normal growth conditions (a), the OxyR protein is inactive. The cI protein binds to R1 and R2 operators, repressing R and stimulating
M . At high concentrations, cI binds also to R3 which caused repression of M . Under oxidative stress conditions (b), OxyR is activated and
binds to the R3 region. This stimulates binding of cI to R1 and R2 , enhancing R repression and M stimulation, and when cI concentration
increases, M repression is prevented by competitive binding to R3 by OxyR. This results in higher activity of M than that under normal
growth conditions, increased levels of cI, and more efficient maintenance of the prophage.
optimal (for prophage induction, i.e., 3 mM) concentration of R2 , and R3 (Figure 3). Binding of the cI protein to R1 and
hydrogen peroxide [32]. Therefore, in H2 O2 -treated lysogenic R2 represses R , the major promoter for expression of genes
bacteria, only a very small fraction of cells (usually less than required during the lytic development, but at the same time
1%) is induced for prophage excision and subsequent lytic stimulates transcription of the cI gene from the M promoter.
development. Since the rest of bacterial population in the At high concentrations of cI, this protein can bind also to
culture can grow and propagate due to resistance to infection R3 which causes a repression of its own promoter M [20]
by the same phage as the prophage present inside the cell, it (Figure 4).
is not possible to observe culture lysis. Detailed molecular studies indicated that OxyR can bind
Another question was what is the mechanism causing specifically to the region of the M -R promoters (introduc-
the low efficiency of prophage induction under conditions of tion of mutations to the putative OxyR-binding site abolished
oxidative stress? Studies on bacteriophage , the best known interactions of this protein with DNA), though with a weaker
representative of lambdoid phages, indicated the factor affinity than to its own promoter [26]. Interestingly, in the
responsible for such a phenomenon. Since DNA sequences presence of OxyR, the cI protein interactions with R1 , R2
of the regulatory regions of and Stx phages are very similar were enhanced, while binding of this repressor to R3 was
[33], one can suppose that the processes occurring in these impaired. These results suggested that OxyR might stimulate
phages are generally the same. repression of R and activation of M but at the same time
It was demonstrated that the prophage induction by downregulate repression of M [26]. This would lead to a
hydrogen peroxide is over 100 times more effective in cells considerably more efficient maintenance of the prophage due
with deletion of the oxyR gene than in wild-type control [26]. to more efficient blocking of the R promoter by abundant
The OxyR protein is a transcription factor acting as a major cI. Indeed, studies with gene fusions showed that while
regulator of the oxidative stress [34]. In the phage DNA region under normal growth conditions (when OxyR is inactive)
responsible for the control of maintenance of the prophage, the activity of the M promoter was similar in both oxyR+
there are 3 sites for binding of the cI repressor, called R1 , and oxyR strains, the oxidative stress conditions (treatment
of cells with H2 O2 which activates OxyR) caused enhanced coli devoid of the stx genes [40] and that bacteriophage-
transcription from M in wild-type bacteria and decreased mediated lysis of STEC is necessary for killing of protist cells
in the oxyR mutant [26]. Therefore, it appears that the by Shiga toxin, since the toxin released as a consequence of
OxyR protein is responsible for the low efficiency of prophage digestion of bacteria by Tetrahymena is harmless to it [41].
induction caused under conditions of the oxidative stress The latter finding was the argument to call the STEC cells a
(Figure 4). Trojan Horse, carrying genes encoding the toxin into target
organisms [42].
3. The Oxidative Stress and Biological Role of
Shiga Toxin Production by STEC 4. Concluding Remarks
There is an intriguing question regarding the biological role The oxidative stress plays a pivotal role in the production of
of Shiga toxin production by STEC strains. As described in Shiga toxins in cells of enterohemorrhagic Escherichia coli
preceding sections, expression of stx genes is effective only (EHEC) infecting human intestine, as well as in response to
after Shiga toxin-converting prophage induction. However, the attack of predator protists. In both cases, hydrogen per-
this also results in subsequent lytic development of the oxide is excreted by eukaryotic cells (either protist predators
bacteriophage and eventual death of the host cell. Thus, or neutrophils in an infected organism) to weaken bacteria
what can be a benefit for the bacterium from production which is a successful strategy against most prokaryotes; how-
of Shiga toxin while it is linked to its death? On the other ever, EHEC strains are lysogenic for Shiga toxin-converting
hand, if toxin production was not beneficial for E. coli, one prophages, and H2 O2 stimulates their induction. This leads
should expect a positive selection of lysogenic bacteria with to the switch to lytic development and production of the
mutations causing deficiency in prophage induction and thus toxin. It appears that Shiga toxin-producing bacteria use the
elimination of STEC cells from the bacterial population. Since specific strategy of bacterial altruism, based on the OxyR-
this is not the case, it should be beneficial for STEC to produce mediated low efficiency of prophage induction during the
Shiga toxin. oxidative stress. As a consequence, only a small fraction of
It was suggested that STEC virulence in humans may be bacterial cells is destroyed due to prophage induction, which
coincidental with the biological role for Shiga toxin being is nevertheless sufficient to produce relatively large amounts
unrelated to human infection [35]. This hypothesis assumed of Shiga toxins able to kill eukaryotic cells. In this way the rest
that synthesis of Shiga toxins by STEC may enhance survival of the E. coli population can survive the attack of the predator
of bacteria in food vacuoles of protozoan predators. Interest- or neutrophils.
ingly, such a phenomenon was demonstrated experimentally
[36]. Moreover, a bacterivorous, protozoan predator, Tetrahy- Conflict of Interests
mena thermophila, was shown to be killed when cocultured The authors declare that there is no conflict of interests
with bacteria lysogenic with Stx bacteriophage [37]. However, regarding the publication of this paper.
this killing did not occur in the presence of catalase, an
enzyme responsible for hydrogen peroxide breakdown [37]. Acknowledgment
In fact, Tetrahymena produces H2 O2 to damage bacterial cells
during attack by this predator [37]. This may be a successful This work was supported by National Science Center
predatory strategy in the case of the vast majority of bacteria; (Poland) Grant no. 2013/09/B/NZ2/02366 to Alicja Wgrzyn.
however, if STEC cells are being attacked, Shiga toxin-
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http://dx.doi.org/10.1155/2016/8453135
Research Article
The Role of the Exo-Xis Region in Oxidative Stress-Mediated
Induction of Shiga Toxin-Converting Prophages
Katarzyna Licznerska,1 Aleksandra Dydecka,1 Sylwia Bloch,1 Gracja Topka,1

Bohena Nejman-FaleNczyk,1 Alicja Wwgrzyn,2 and Grzegorz Wwgrzyn1
1
Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
2
Laboratory of Molecular Biology (Affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdansk, Poland
Correspondence should be addressed to Grzegorz Wgrzyn; grzegorz.wegrzyn@biol.ug.edu.pl
Received 11 August 2015; Revised 22 September 2015; Accepted 28 September 2015
Copyright 2016 Katarzyna Licznerska et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction
processes. The effects were dramatic in Shiga toxin-converting phage 24B after treatment with oxidative stress-inducing agent,
hydrogen peroxide, while they were less pronounced in bacteriophage and in both phages irradiated with UV. The hydrogen
peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells
lysogenic for either or 24B , deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the
S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for
lytic development (particularly xis, exo, N, cro, O, Q, and R) could be observed in 24B -, but not in -bearing cells. We conclude
that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and
regulatory genes of Shiga toxin-converting bacteriophage 24B .
1. Introduction [12]. stx genes are present between antiterminator gene and
the genes coding for proteins causing cell lysis; thus, in the
Infection of humans by enterohemorrhagic Escherichia coli lysogenic state, these genes are not transcribed [1315] and
(EHEC) strains causes hemorrhagic colitis, and in some Shiga toxins are not produced. Their expression is possible
patients it may result in various complications, including, the only after prophage induction [11, 12] which usually requires
most severe of them, the hemolytic-uremic syndrome and activation of the bacterial S.O.S. response, mediated by RecA
neurological dysfunctions [13]. The main causes of EHEC- protein, though RecA-independent induction of Shiga toxin-
mediated complications are Shiga toxins, produced by the converting prophages by EDTA has also been reported [16].
infecting bacteria [4]. The severity of EHEC infections and During infection of human intestine by EHEC, the oxida-
significance of the medical problem related to them are exem- tive stress appears to be the most likely condition causing the
plified by local outbreaks, occurring in various geographical bacterial S.O.S. response and subsequent induction of Shiga
regions around the world. One of the most famous of them toxin-converting prophages [17]. In fact, it was demonstrated
took place in 2011 in Germany, where over 4,000 patients that hydrogen peroxide (which is produced by neutrophils as
developed severe symptoms, and 54 died [510]. a response to infection) enhanced production of Shiga toxins
In EHEC strains, Shiga toxins are encoded by genes by EHEC [18] due to oxidative stress-mediated induction of
(called stx genes) located in genomes of prophages [11, 12]. Shiga toxin-converting prophages [19, 20].
The phages bearing stx genes are referred to as Shiga toxin- Since many antibiotics not only kill bacteria and inhibit
converting bacteriophages, and all of them belong to the fam- their growth but also induce prophage lytic development,
ily of lambdoid phages (with phage serving as a paradigm) their use is not recommended when EHEC infection is
pL
orf60a
orf55
orf61
ea8.5
ea22
cIII
exo

xis
N
tL
orf63
orf73
pL
Putative C4 zinc
finger protein
vB_24_1c
vB_24_3c
vB_24_4c
vB_24_8c
24B
cIII
exo
xis
N
tL
vB_24_7c
vB_24_2c
vB_24_6c
vB_24_9c
vB_24_5c
Figure 1: Schematic maps of the exo-xis regions of bacteriophages and 24B . Genes from the exo-xis region are marked as thick grey
arrows, and other genes are shown as thick open arrows. Directionality of transcription from promoter is indicated as thin dashed arrow.
1 terminator is marked as black oval.
confirmed or even suspected (reviewed in [12]). Therefore, medium at 30 C (most experiments) or 37 C (lysogenization
there are serious problems with treatment of patients, indi- and recombination procedures during construction of strains
cating that searching for new targets of potential therapies and SOS ChromoTest, according to the instructions of kits
against Shiga toxin-producing bacteria is important. One manufacturers), under aerobic conditions. Where appropri-
might consider that such therapies should be focused on ate, the following antibiotics were added: chloramphenicol up
inhibition of Shiga toxin-converting prophage induction to 20 g/mL, kanamycin up to 50 g/mL, and/or tetracycline
which would impair production of the EHEC virulence fac- up to 12.5 g/mL.
tor. All lambdoid phages, including Shiga toxin-converting Bacteriophages papa (from our collection) [26] and
bacteriophages, contain the region in their genomes which 24B (stx2::cat) [28] were employed in this study. Phage
is dispensable for the development under standard laboratory suspensions were stored in the TM buffer (10 mM Tris-HCl,
conditions [14, 15]. Inside this part of the phage genome, there 10 mM MgSO4 , pH 7.2) at 4 C.
is an evolutionarily conserved fragment, located between exo The deletion mutants were constructed as described
and xis genes and transcribed from promoter, called the previously [29], by using the Quick and Easy E. coli Gene
exo-xis region (Figure 1). This region encompasses several Deletion Kit (from Gene Bridges). The deletion of the indi-
genes and open reading frames whose functions in phage cated region was performed according to the manufacturers
development are largely unknown, and only a few articles are protocol using primers listed in Table 2. In the first step, the
available in the literature that focused on them. Nevertheless, targeted sequence has been replaced with the FRT-flanked
some interesting observations have been reported. Namely, kanamycin resistance cassette, and the selection marker was
induction of expression of genes from the exo-xis region subsequently removed in the FLP-recombinase step, leaving
resulted in synchronization of the host cell cycle [21] and only 87 nucleotides of the cassette in the place of the original
inhibition of host DNA replication [22]. Moreover, overex- sequence. Each deletion was confirmed by DNA sequencing.
pression of these genes impaired lysogenization of E. coli by Lysogenic strains were constructed according to the pro-
bacteriophage [23] and enhanced induction of prophages cedure described previously [24], with slight modifications.
and 24B (one of Shiga toxin-converting phages) [24]. Briefly, host bacteria were cultured to A600 of 0.5 in LB
Ea8.5 protein, encoded by a gene located in the exo-xis medium supplemented with MgSO4 and CaCl2 (to final
region, contains a fused homeodomain/zinc finger fold [25] concentrations of 10 mM each) at 37 C with shaking. At
which suggests a regulatory role for this protein. Interestingly, this point, one milliliter of the culture was withdrawn and
prophage induction with mitomycin C or hydrogen peroxide centrifuged (10 min, 2000 g). Pellet was washed twice with
caused different expression patterns of genes from the exo- TCM buffer (10 mM Tris-HCl, pH 7.2, 10 mM MgSO4 , 10 mM
xis region; such differences were observed in both phages, CaCl2 ) and then suspended in 1 mL of the same buffer. Next,
and 24B [26]. In this work, we used the deletion mutants to bacteria were incubated for 30 min at 30 C and mixed with
investigate the role of the exo-xis region in induction of and phage suspensions at multiplicity of infection (m.o.i.) = 5.
24B prophages under oxidative stress conditions. Mixtures of bacteria and phages were incubated in TMC
buffer for 30 min at 30 C; then serial dilutions were prepared
2. Materials and Methods in TM buffer (10 mM Tris-HCl, 10 mM MgSO4 ; pH 7.2), and
the mixture was plated onto LB agar. Plates were incubated at
2.1. Bacteria and Bacteriophages. E. coli MG1655 strain [27] 37 C overnight. Lysogens were verified by sensitivity to UV
and its derivatives, used in this work, are listed in Table 1. irradiation and confirmed by PCR with primers designed to
Bacteria were routinely cultured in the Luria-Bertani (LB) amplify phage sequence (Table 3).
Table 1: Escherichia coli strains.

Strain Genotype or relevant characteristics Reference
E. coli MG1655 F ilvG rfb-50 rph-1 [27]
E. coli MG1655 () MG1655 bearing prophage [24]
E. coli MG1655 (exo-xis) MG1655 bearing prophage with deletion of all orfs and genes This study, by recombination
localized between genes exo and xis
E. coli MG1655 (orfs) MG1655 bearing prophage with deletion of orf60a, orf63, This study, by recombination
orf61, and orf73
E. coli MG1655 (orf60a) MG1655 bearing prophage with deletion of orf60a This study, by recombination
E. coli MG1655 (orf63) MG1655 bearing prophage with deletion of orf63 This study, by recombination
E. coli MG1655 (ea22) MG1655 bearing prophage with deletion of ea22 This study, by recombination
E. coli MG1655 (ea8.5) MG1655 bearing prophage with deletion of ea8.5 This study, by recombination
E. coli MG1655 (24B ) MG1655 bearing 24B prophage [24]
E. coli MG1655 (24B exo-xis) MG1655 bearing 24B prophage with deletion of all orfs and This study, by recombination
genes localized between genes exo and xis
E. coli MG1655 (24B orfs) MG1655 bearing 24B prophage with deletion of 4 orfs being This study, by recombination
homologues of orf60a, orf63, orf61, and orf73
E. coli MG1655 (24B orf60a) MG1655 bearing 24B prophage with deletion of vb 24B 9c, the This study, by recombination
homologue of orf60a
E. coli MG1655 (24B orf63) MG1655 bearing 24B prophage with deletion of vb 24B 8c, the This study, by recombination
homologue of orf63
E. coli MG1655 (24B orf61) MG1655 bearing 24B prophage with deletion of vb 24B 7c, the This study, by recombination
homologue of orf61
E. coli MG1655 (24B orf73) MG1655 bearing 24B prophage with deletion of the sequence This study, by recombination
of putative C4 zinc finger protein, the homologue of orf73
E. coli MG1655 (24B ea22) MG1655 bearing 24B prophage with deletion of vb 24B 6c, the This study, by recombination
analogue of ea22
E. coli MG1655recA13 MG1655 but recA13 [33]
E. coli MG1655recA13 () MG1655recA13 bearing prophage This study, by lysogenization
E. coli MG1655recA13 (exo-xis) MG1655recA13 bearing prophage with deletion of all orfs and This study, by lysogenization
genes localized between genes exo and xis
E. coli MG1655recA13 (24B ) MG1655recA13 bearing 24B prophage This study, by lysogenization
E. coli MG1655recA13 (24B exo-xis) MG1655recA13 bearing 24B prophage with deletion of all orfs This study, by lysogenization
and genes localized between genes exo and xis
E. coli PQ37 sfiA::Mud(Ap lac) cts, lacU169, mal+ , galE, galY, PhoC, rfa, F , [31]
thr, leu, his, pyrD, thi, trp::MUC+ , srl300::Tn10, rpoB, uvrA+
E. coli PQ37 () PQ37 bearing prophage This study, by lysogenization
E. coli PQ37 (exo-xis) PQ37 bearing prophage with deletion of all orfs and genes This study, by lysogenization
E. coli PQ37 (24B ) PQ37 bearing 24B prophage This study, by lysogenization
E. coli PQ37 (24B exo-xis) PQ37 bearing 24B prophage with deletion of all orfs and genes This study, by lysogenization
2.2. Phage Lytic Development after Prophage Induction. Bac- Then, serial dilutions were prepared in TM buffer, and
teria lysogenic with tested phages were cultured in LB phage titer (number of phages per mL) was determined by
medium at 30 C to A600 of 0.1. Two induction agents were spotting 2.5 L of each dilution of the phage lysate on a
tested: H2 O2 (1 mM) and UV irradiation (50 J/m2 ; this dose freshly prepared LB agar (1.5%) or LB agar with 2.5 g/mL
was achieved by 20 sec incubation of the bacterial suspen- chloramphenicol (according to a procedure described previ-
sions in Petri dishes under UV lamp hanged 17 cm above ously [30]), with a poured mixture of 1 mL of the indicator
the laboratory table). At indicated times after induction, E. coli MG1655 strain culture and 2 mL of 0.7% nutrient
samples of bacterial cultures were harvested, and 30 L of agar (prewarmed to 45 C), supplemented with MgSO4 and
chloroform was added to 0.5 mL of each sample. The mixture CaCl2 (to a final concentration of 10 mM each). When full-
was vortexed and centrifuged for 5 min in a microcentrifuge. plate titration was used, 0.1 mL of phage lysate dilutions was
4
Table 2: Primers used for construction of E. coli strains.

Primer name Sequence (5 3 )
pF exo-xis = pF orf60a ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
pR exo-xis AGGCGGCTTGGTGTTCTTTCAGTTCTTCAATTCGAATATTGGTTACGTCTTAATACGACTCACTATAGGGCTC
pF orfs = pF orf60a ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
pR orfs = pR orf73 ACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAGTAATACGACTCACTATAGGGCTC
pF orf60a ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
pR orf60a AGATGCTTTGTGCATACAGCCCCTCGTTTATTATTTATCTCCTCAGCCAGTAATACGACTCACTATAGGGCTC
pF orf63 CACAAAGCATCTTCTGTTGAGTTAAGAACGAGTATCGAGATGGCACATAGAATTAACCCTCACTAAAGGGCG
pR orf63 TCATACCTGGTTTCTCTCATCTGCTTCTGCTTTCGCCACCATCATTTCCATAATACGACTCACTATAGGGCTC
pF orf61 AGAGAAACCAGGTATGACAACCACGGAATGCATTTTTCTGGCAGCGGGCTAATTAACCCTCACTAAAGGGCG
pR orf61 TTATCCGGAAACTGCTGTCTGGCTTTTTTTGATTTCAGAATTAGCCTGACTAATACGACTCACTATAGGGCTC
pF orf73 ACATCATTGATTCAGCATCAGAAATAGAAGAATTACAGCGCAACACAGCAAATTAACCCTCACTAAAGGGCG
pR orf73 ACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAGTAATACGACTCACTATAGGGCTC
pF ea22 GAAATTAACTCTCAGGCACTGCGTGAAGCGGCAGAGCAGGCAATGCATGAAATTAACCCTCACTAAAGGGCG
pR ea22 GTCAGACATCATATGCAGATACTCACCTGCATCCTGAACCCATTGACCTCCTAATACGACTCACTATAGGGCTC
pF ea8.5 ATGAGTATCAATGAGTTAGAGTCTGAGCAAAAAGATTGGGCGTTATCAATAATTAACCCTCACTAAAGGGCG
pR ea8.5 TAATCATCTATATGTTTTGTACAGAGAGGGCAAGTATCGTTTCCACCGTATAATACGACTCACTATAGGGCTC
pF 24B exo-xis = pF 24B orf60a TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
pR 24B exo-xis TGGCAATATGCTTTTCCTTCTCAATTTCCGCTTTAATCATATGCAGTTCGTAATACGACTCACTATAGGGCTC
pF 24B orfs = pF 24B orf60a TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
pR 24B orfs = pR 24B orf73 CTTCGAACCTCTCTGTTTACTGATAAGCTCCAGATCCTCCTGGCAACTTGTAATACGACTCACTATAGGGCTC
pF 24B orf60a TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
pR 24B orf60a GGAGATGCTTTGTGCATACAGCCCCTCGTTATTATTTATCTCTTCAGCCTAATACGACTCACTATAGGGCTC
pF 24B orf63 TGCACAAAGCATCTCCTGTTGAATTAAGAACGAGTATCGGGATGGCACATAATTAACCCTCACTAAAGGGCG
pR 24B orf63 CATCATTTCCAGCTTTTGTGAAAGGGATGTGGCTAACGTATGAAATTCTTTAATACGACTCACTATAGGGCTC
pF 24B orf61 TTCTGGCAGCAGGCTTCATATTCTGTGTGCTTATGCTTGCCGACATGGGAAATTAACCCTCACTAAAGGGCG
pR 24B orf61 CACCGTTCCTTAAAGACGCCGTTTAACATGCCGATCGCCAGACTTAAATGTACGACTCACTATAGGGCTC
pF 24B orf73 TGGCAGACCTCATTGATTCAGCATCAGAAATTGAAGAATTACAGCGCAACAATTAACCCTCACTAAAGGGCGG
pR 24B orf73 CTTCGAACCTCTCTGTTTACTGATAAGCTCCAGATCCTCCTGGCAACTTGTAATACGACTCACTATAGGGCTC
pF 24B ea22 ATCAGGCACTGCGTGAAAAGGCAGAGAAAGCAACTAAAGGAAGCTACATCAATTAACCCTCACTAAAGGGCG
pR 24B ea22 ATCCAGTGTGACGGTTTCCACGACGCACCAGGAATTATCCACCCATCATTATACGACTCACTATAGGGCTC
Table 3: Primers used for PCR.

Primers for bacterial sequences Primers for phage sequences
Name Sequence (5 3 ) Name Sequence (5 3 )
pF recA AGATTTCGACGATACGGCCC pF int TTTGATTTCAATTTTGTCCCACT
pR recA AACCATCTCTACCGGTTCGC pR int ACCATGGCATCACAGTATCG
pF lexA ATGGATGGTGACTTGCTGGC pF xis TACCGCTGATTCGTGGAACA
pR lexA TTCGTCATCAATACGTGCGAC pR xis GGGTTCGGGAATGCAGGATA
pF ssb ATCGAAGGTCAGCTGCGTAC pF exo TGCCGTCACTGCATAAACC
pR ssb CGACTTCTGTGGTGTAGCGA pR exo TCTATCGCGACGAAAGTATGC
pF recF CGATACCGGCGCTATACTCC pF cIII ATTCTTTGGGACTCCTGGCTG
pR recF TTACGAACAGCTACGCCCG pR cIII GTAAATTACGTGACGGATGGAAAC
pF rpoD GAATCTGAAATCGGGCGCAC pF N CTCGTGATTTCGGTTTGCGA
pR rpoD GTCAACAGTTCAACGGTGCC pR N AAGCAGCAAATCCCCTGTTG
pF rpoH GCTTTGGTGGTCGCAACTTT pF cI ACCTCAAGCCAGAATGCAGA
pR rpoH TCGCCGTTCACTGGATCAAA pR cI CCAAAGGTGATGCGGAGAGA
pF rpoS TTGCTCTGCGATCTCTTCCG pF cro ATGCGGAAGAGGTAAAGCCC
pR rpoS GAACGTTTACCTGCGAACCG pR cro TGGAATGTGTAAGAGCGGGG
pF uvrA GTCCATATCCGCCACTACCG pF cII TCGCAATGCTTGGAACTGAGA
pR uvrA TTACCCAACGTCTTGCCGAG pR cII CCCTCTTCCACCTGCTGATC
pF ftsK ACAAACCGTTTATCTGCGCG pF O AATTCTGGCGAATCCTCTGA
pR ftsK ATCTTTACCCAGCACCACGG pR O GAATTGCATCCGGTTT
pF 16SrRNA CCTTACGACCAGGGCTACAC pF Q TTCTGCGGTAAGCACGAAC
pR 16SrRNA TTATGAGGTCCGCTTGCTCT pR Q TGCATCAGATAGTTGATAGCCTTT
pF R ATCGACCGTTGCAGCAATA
pR R GCTCGAACTGACCATAACCAG
pF 24B int CAGTTGCCGGTATCCCTGT
pR 24B int TGAGGCTTTCTTGCTTGTCA
pF 24B xis TATCGCGCCGGATGAGTAAG
pR 24B xis CGCACAGCTTTGTATAATTTGCG
pF 24B exo TGCCGTCACTGCATAAACC
pR 24B exo TCTATCGCGACGAAAGTATGC
pF 24B cIII ATTCTTTGGGACTCCTGGCTG
pR 24B cIII GTAAATTACGTGACGGATGGAAAC
pF 24B N AGGCGTTTCGTGAGTACCTT
pR 24B N TTACACCGCCCTACTCTAAGC
pF 24B cI TGCTGTCTCCTTTCACACGA
pR 24B cI GCGATGGGTGGCTCAAAATT
pF 24B cro CGAAGGCTTGTGGAGTTAGC
pR 24B cro GTCTTAGGGAGGAAGCCGTT
pF 24B cII TGATCGCGCAGAAACTGATTTAC
pR 24B cII GACAGCCAATCATCTTTGCCA
pF 24B O AAGCGAGTTTGCCACGAT
pR 24B O GAACCCGAACTGCTTACCG
pF 24B Q GGGAGTGAGGCTTGAGATGG
pR 24B Q TACAGAGGTTCTCCCTCCCG
pF 24B R GGGTGGATGGTAAGCCTGT
pR 24B R TAACCCGGTCGCATTTTTC
plated onto LB agar. Plates were incubated at 37 C overnight. forming units (pfu)/mL, was calculated by subtracting the
Analogous experiment but without induction agents (con- values obtained in the control experiment from the values
trol experiments), which allows estimation of effects of determined in the main experiment, and as a consequence
spontaneous prophage induction, was performed with each it represents the ratio of phage titers in induced and nonin-
lysogenic strain. The relative phage titer, expressed as plaque duced cultures. Each experiment was repeated three times.
2.3. The S.O.S. Assay. The S.O.S. assay was performed using Master Mix, 6.25 ng/L cDNA, and 200 nM of each gene-
the SOS-ChromoTest Kit (Environmental Bio-Detection specific primer (Table 3). Relative quantification assays were
Products Inc.), following the manufacturers protocol and performed with cDNA of 16S rRNA and phage/bacterial
using provided 4-nitro-quinoline oxide (4-NQO) as a posi- genes multiplex assay. All templates were amplified using the
tive reference standard, and 1 mM H2 O2 and UV irradiation following program: incubation at 95 C for 5 min, followed by
(50 J/m2 ) as tested inducers of the S.O.S. response [31, 32]. 55 cycles of 95 C for 10 s, 60 C for 15 s, and 72 C for 15 s. No
In the case of UV light irradiation, the production of - template control was included with each run. The specificity
galactosidase was evaluated immediately after the expo- of amplified products was examined by melting curve analysis
sure, without 2 h incubation at 37 C (recommended by immediately after the final PCR cycle and confirmed by gel
the manufacturer), as, without this modification, the visual electrophoresis. Each experiment was conducted in triplicate.
detection of the blue color was not possible due to rapid The relative changes in gene expression revealed by quan-
S.O.S. response after UV irradiation. Before use, the SOS- titative real-time PCR experiments were analyzed using the
ChromoTest bacterial strain (E. coli PQ37, provided with the calibrator, normalizing relative quantification method with
kit) was lysogenized by following phages: , exo-xis, 24B , efficiency correction (called the E-Method). This method has
or 24B exo-xis, according to procedure described above. been used and described in detail previously [26, 29, 35].
The values obtained at time 0 (representing the conditions
of spontaneous prophage induction) were used as calibrators.
2.4. Preparation of RNA and cDNA from Bacteria. For the
Thus, the following equation was employed to calculate the
isolation of total RNA, the previously described [26] pro-
final results:
cedure was employed. Briefly, the prophage induction was
performed with 1 mM H2 O2 or UV irradiation (at the dose of Normalized relative ratio
50 J/m2 ). Following induction, the samples were withdrawn
at indicated times and the growth of bacteria was inhibited by CT() calibratorCT() sample (1)
the addition of NaN3 (Sigma-Aldrich) to a final concentration = CT() calibratorCT() sample
,

of 10 mM. Total RNA was isolated from 109 bacterial cells
by using the High Pure RNA Isolation Kit (Roche Applied where is target and is reference.
Science). Bacterial genomic DNA carryover was removed by
incubation with TURBO DNase from TURBO DNA-free Kit 2.6. In Silico Analyses. The multiple sequence alignment was
(Life Technologies) for 60 min at 37 C, according to the man- performed using the ClustalW algorithm available at the web-
ufacturers guidelines. To evaluate the quality and quantity site http://www.genome.jp/tools/clustalw/. The Pfam protein
of the isolated RNA, a NanoDrop spectrophotometer was families database [36], available at the website http://pfam
employed, considering the absorbance ratio (which should .xfam.org/, was used to identify protein domains.
be 1.8 A260/A280 2.0). Moreover, band patterns of total
RNA were visualized by electrophoresis. The absence of DNA
from RNA samples was controlled by PCR amplification 3. Results and Discussion
and by real-time PCR amplification (all analyzed genes were 3.1. Deletion of the Exo-Xis Region Impairs 24B but Not
tested). RNA preparations were stored at 80 C. cDNA Prophage Induction after Treatment with Hydrogen Peroxide.
was obtained with Transcriptor Reverse Transcriptase and Until now, all in vivo studies on effects of the exo-xis region
random hexamer primers (Roche Applied Science), using on host or phage development were performed with the use
total RNA samples (1.25 g) as templates. cDNA reaction of strains overexpressing genes from this region [2124, 26].
mixtures were diluted 10-fold for use in real-time PCR. In this work, we have constructed a series of bacteriophage
and 24B mutants with deletions of either the whole exo-xis
2.5. Real-Time PCR Assay and Data Analysis. The patterns of region or individual genes or open reading frames (Table 1).
genes expression were determined by quantitative real-time When wild-type and 24B prophages were induced by UV
reverse transcription-PCR (qRT-PCR), using the LightCycler irradiation (employed in this work as positive control condi-
480 Real-Time PCR System (Roche Applied Science) and tions causing effective prophage induction) or hydrogen per-
cDNA samples from lysogenic bacteria. Transcripts of tested oxide treatment of the lysogenic cells, efficiencies of induction
phage and bacterial genes were compared in parallel to and further phage lytic development were comparable in
16S rRNA housekeeping gene (according to a procedure both phages, though some differences were observed in the
described previously [34]), whose expression was found to duration of the lag phase of the phage development (Figure 2).
be constant. Primers were developed by Primer3web version Induction of exo-xis mutant with UV irradiation was
4.0.0 and produced by Sigma-Aldrich or GENOMED. The similar to that observed for the wild-type , and treatment
transcriptional analysis of phage and bacterial genes from with hydrogen peroxide caused only a slight delay in the
lysogenic strains was performed with primers presented mutant phage development. The decrease in the phage titer
in Table 3. Real-time PCR amplifications were carried out at later times of the experiments is characteristic for and
for 55 cycles in 20 L reaction volume, using LightCycler most probably arises from adsorption of the progeny virions
480 SYBR Green I Master (Roche Applied Science) as on fragments of disrupted cell envelopes [15, 24]. However,
a fluorescent detection dye. Reactions were performed in induction of 24B exo-xis prophage by UV irradiation was
Roche 96-well plates containing 10 L 2x SYBR Green I less efficient than that of the wild-type 24B , and induction
24B
9 9
10 10
108
Relative phage titer (PFU/mL)

108
Relative phage titer (PFU/mL)
10 7 107
10 6 106
10 5 105
10 4 104
10 3 103
10 2 102
10 1 101
10 0 100
0 60 120 180 240 300 360 0 60 120 180 240 300 360
Time (min) Time (min)
(a) (b)
Figure 2: Lytic development of bacteriophages (a) and 24B (b), either wild-type (open symbols) or exo-xis (closed symbols), after
induction of lysogenic E. coli MG1655 with UV irradiation (50 J/m2 , circles) or hydrogen peroxide (1 mM, triangles). The presented results are
mean values from 3 independent experiments (biological samples), with error bars indicating SD. Statistically significant differences ( < 0.05
in t-test) between wild-type and exo-xis phages were found at times 270, 300, and 360 min of experiments with hydrogen peroxide and at
270, 300, 330, and 360 min of experiments with UV for and at times 120, 240, 270, 300, 330, and 360 min of experiments with hydrogen
peroxide and at 270, 300, 330, and 360 min of experiments with UV for 24B .
LexA DNA binding domain/HTH3 domain

LexA ----MKALTARQQEVFDLIRDHISQTG--MPPTRAEIAQRLGFRSP---- 40
cI MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSGVGA 50
cI 24B ----MVQNEKVRKEFAQRLAQACKEAGLDEHGRGMAIARALSLSSKGVSK 46
: : .: : : . : .
LexA -----NAAEEH----LKALARKGVIEIVSGASRGIRLLQE---------E 72
cI LFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRS 100
cI 24B WFNAESLPRQEKMNALAKFLNVDVVWLQHGTSLNGANDEDTLS----FVG 92
. . : . . : . : :
LexA EEGLPLVGRVAAG---EPLLAQQHIEGHYQVDPSLFKPNADFLLRVSGMS 119

cI EYEYPVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNS 150
cI 24B KLKKGLVRVVGEAILGVDGAIEMTEERDGWLKIYSDDPD-AFGLRVKGDS 141
: :. . : . :. . : . .
Peptidase S24 domain
LexA MKDIG-----IMDGDLLAVHKTQDVRNGQVVVARID-DEVTVKRLKKQ-G 162

cI MTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRD-S 199
cI 24B MWPRIK------SGEYVLIEPNTKVFPGDEVFVRTVEGHNMIKVLGYDRD 185
. : : . : . . . . . : .
LexA NKVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL------------ 202

cI GQVFLQPLNPQYPMIPCN---ESCSVVGKVIASQWPEETFG----------- 237
cI 24B GEYQFTSINQDHRPITLP--YHQVAKVEYVAGILKQSRHLDDIEAREWLKSS 235
.: : . :. .. : . . :
Figure 3: Alignment of amino acid sequences of E. coli LexA protein and cI repressors of bacteriophages and 24B . Specific protein domains
are indicated by grey background. Self-cleavage sites are underlined (two amino acid residues between which the cleavage occurs). The active
sites of the peptidase domains are framed. Symbols under the sequence alignment indicate conserved sequence (), conservative mutations
(:), semiconservative mutations (.), and nonconservative mutations ().
of the mutant by hydrogen peroxide was severely impaired peroxide was at the same range (103 /mL) as that measured
(Figure 2). More detailed analyses, based on the full-plate without specific induction (i.e., representing efficiency of
phage titration method, allowing detection of 10 pfu/mL (see spontaneous prophage induction). Nevertheless, the titer of
Section 2 for details), indicated that the number of pfu per 24B exo-xis measured at 240 and 360 min after induction
mL of 24B exo-xis phage after induction with hydrogen was 9.0 0.2 103 and 7.9 0.9 103 , respectively, that
No inductor
UV light
4-NQO
H 2 O2
195 123 171 -Galactosidase activity

Alkaline phosphatase activity
exo-xis 202 119 140 -Galactosidase activity

24B

24B exo-xis
Figure 4: Induction of the S.O.S. response in E. coli PQ37 lysogenic with , exo-xis, 24B , or 24B exo-xis, treated with 4-NQO (4-
nitro-quinoline oxide, positive control), H2 O2 (1 mM), or UV (50 J/m2 ), using the SOS ChromoTest. -Galactosidase activity (identified
by the blue spots) represents induction of the S.O.S. regulon. Alkaline phosphatase activity (identified by yellow spots) evaluates viability
of tested bacteria. Quantification of -galactosidase activity was performed by densitometry, using the ImageJ software (available at
http://imagej.nih.gov/ij/index.html). The results (in arbitrary units reflecting value = 1 ascribed to samples with no inductor), presented
as numbers inside the corresponding spots, are mean values from three measurements (with SD < 10% in each case). All these values were
significantly ( < 0.001 in t-test) higher than that in the control experiments with no inductor. When exo-xis mutants were compared
to wild-type phages, the only significant difference ( < 0.05 in t-test) occurred between 24B and 24B exo-xis lysogens induced with
hydrogen peroxide.
is, still 3-4 times higher than that without induction, which the oxidative stress, the most likely conditions causing Shiga
was 2.1 0.3 103 and 2.6 0.4 103 , respectively (note toxin-converting prophage induction during infection with
that the titer of the wild-type 24B after prophage induction EHEC.
was several orders of magnitude higher than that without
induction, Figure 2). 3.2. Hydrogen Peroxide-Mediated Prophage Induction Is a
Deletions of individual genes and open reading frames RecA-Dependent Process. Efficient induction of lambdoid
from the exo-xis region in 24B did not affect significantly prophages is a RecA-dependent process due to a molecular
the phage titer. However, such deletions resulted in delays in mimicry between the phage cI repressor and the host-
prophage induction by hydrogen peroxide (Table 4). Interest- encoded LexA repressor which is self-cleaved after stimula-
ingly, when prophage induction was stimulated by UV irra- tion by the activated form of RecA protein under the S.O.S.
diation, such effect was not observed, and in some cases even response conditions [1215]. Such a mimicry is well known
more rapid induction of the mutant prophages occurred. In for bacteriophage cI protein and LexA [12, 13], and we
bacteriophage , only slight effects of deletions of individual found that both domain structure and amino acid residues
genes and open reading frames were detected (Table 4). crucial for the self-cleavage are also conserved in cI repressor
We conclude that the genes and open reading frames of phage 24B (Figure 3) (note that cI sequence of 24B is
from the exo-xis region play important roles in the regulation identical to that of another Shiga toxin-converting bacterio-
of lambdoid prophage induction, as deletions of the whole phage, 933 W [37]). Nevertheless, since RecA-independent
region or single loci caused significant changes in efficiency induction of Shiga toxin-converting prophages has also been
and timing of this process. The effects of mutations are more reported [16], we asked whether hydrogen peroxide-caused
pronounced in Shiga toxin-converting phage 24B than in prophage induction depends on the activation of the S.O.S.
and in lysogenic E. coli cells treated with hydrogen peroxide response.
than in UV-irradiated ones. Thus, the exo-xis region seems to When testing H2 O2 - or UV-dependent induction of
be particularly important for 24B phage under conditions of prophages , exo-xis, 24B , and 24B exo-xis in recA13
40
35
30
25
20
15
E. coli () 10
20 E. coli (24B ) 5
Relative quantification results
18 20 0

16 18 recA lexA ssb recF rpoD rpoH rpoS uvrA ftsK
14 16
(E-Method)
12 14
(E-Method)
10 12
8 10
6 8
4 6
2 4
0 2
recA lexA ssb recF rpoD rpoH rpoS uvrA ftsK 0
recA lexA ssb recF rpoD rpoH rpoS uvrA ftsK
4 min 32 min
8 min 64 min 4 min 32 min
20 min 16 min 80 min
20 min
E. coli (exo-xis) E. coli (24B exo-xis)

20 20
18 18
16 16
14 14
(E-Method)
(E-Method)
12 12
10 10
8 8
6 6
4 4
2 2
0 0
recA lexA ssb recF rpoD rpoH rpoS uvrA ftsK recA lexA ssb recF rpoD rpoH rpoS uvrA ftsK

20 min 20 min
Figure 5: Expression of genes from the S.O.S. regulon in E. coli MG1655 lysogenic with , exo-xis, 24B , or 24B exo-xis, at indicated
times after treatment with 1 mM H2 O2 , as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated
cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate
the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with
error bars indicating SD. The additional panel for E. coli (24B ) represents the results with different scale.
mutant host, in the assays analogous to those presented in was considerably weaker than in the analogous experiment
Figure 2, pfu/mL values were at the levels of those estimated with 24B lysogen (Figure 4). No such difference could be
for the uninduced controls (1.8 0.1 102 , 4.1 1.4 102 , observed in UV-irradiated bacteria (Figure 4).
1.3 0.1 103 , and 2.8 0.3 103 pfu/mL for , exo-
xis, 24B , and 24B exo-xis, resp.). Therefore, we conclude 3.3. Deletion of the Exo-Xis Region Negatively Influences
that induction of the investigated prophages under conditions Expression of Genes from the S.O.S. Regulon in Hydrogen
of the oxidative stress (treatment with hydrogen peroxide) Peroxide-Treated Lysogenic Bacteria. Since unexpected
strongly depends on RecA function. Indeed, in cells lysogenic results were obtained in experiments with hydrogen
for or 24B and treated with UV light or hydrogen perox- peroxide-treated 24B exo-xis lysogenic cells (Figure 4),
ide, efficient induction of the S.O.S. response was evident, as we aimed to investigate the phenomenon of a less efficient
estimated with the SOS ChromoTest (Figure 4). Intriguingly, induction of the S.O.S. response in more detail. Thus,
while induction of the S.O.S. response by hydrogen peroxide expression of genes from the S.O.S. regulon was tested by
in exo-xis lysogen was comparable to that in lysogen, reverse transcription quantitative real-time PCR in E. coli
the signal in the SOS ChromoTest in 24B exo-xis lysogen cells lysogenic for , exo-xis, 24B , and 24B exo-xis
12
10
8
E. coli () E. coli (24B ) 6
60 300 4


50 250 0
int xis exo cIII N cI cro cII O Q R
(E-Method)
40 200
(E-Method)
30 150

20 100

10 50

0 0
int xis exo cIII N cI cro cII O Q R int xis exo cIII N cI cro cII O Q R
exo- xis + exo- xis +

exo-xis exo-xis
Figure 6: Expression of selected bacteriophage genes in E. coli MG1655 lysogenic with or 24B , either wild-type (blue columns) or exo-xis
(yellow columns) at 160 min after treatment with 1 mM H2 O2 , as estimated by reverse transcription quantitative real-time PCR. The values
obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the
presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments
(biological samples), with error bars indicating SD. The additional panel for E. coli (24B ) represents the results of exo-xis variant with
different scale, due to very small values measured. Statistically significant differences (in t-test) are marked as follows: < 0.05, < 0.01,
and < 0.001.
Table 4: Duration of the lag phase of the phage lytic development gene in both phages and ssb, uvrA, and ftsK genes in 24B ,
after prophage induction with either hydrogen peroxide (1 mM) or especially at later times after the treatment (Figure 5).
UV irradiation (50 J/m2 ). Interestingly, in the case of wild-type 24B lysogenic cells,
The time range of the switch from lag to the enhanced expression of particular genes from the S.O.S.
Strain log phase regulon persisted longer, in most cases until 16 min after
H2 O2 (1 mM) UV (50 J/m2 ) induction, whereas in the deletion mutant it decreases after
MG1655 () 6090 min 60 min 4 min (Figure 5). The impairment in expression of genes
MG1655 (exo-xis) 90120 min 3060 min from the S.O.S. regulon (in particular recA and lexA genes,
MG1655 (orfs) 6090 min 3060 min encoding the main regulators of the S.O.S. response) in the
MG1655 (orf60a) 6090 min 3060 min absence of the exo-xis region was more pronounced in 24B
MG1655 (orf63) 6090 min 3090 min than in . Moreover, induction of the S.O.S. regulon occurred
MG1655 (orf61) 6090 min 3060 min significantly earlier in 24B and 24B exo-xis lysogens than
MG1655 (orf73) 3060 min 030 min in cells bearing and exo-xis prophages (Figure 5). These
MG1655 (ea22) 6090 min 3060 min results might explain, at least partially, effects of deletions
MG1655 (ea8.5) 6090 min 3060 min of exo-xis genes on prophage induction, demonstrated in
MG1655 (24B ) 6090 min 150180 min Figure 2 and Table 4, particularly delayed induction of 24B
MG1655 (24B exo-xis) a 150180 min prophage devoid of certain genes and open reading frames,
MG1655 (24B orfs) 150180 min 150180 min and less pronounced effects of their lack in than in 24B .
MG1655 (24B orf60a) 120150 min 90120 min Indications that overexpression of some genes from
MG1655 (24B orf63) 120150 min 3060 min the exo-xis region of can influence host cell cycle and
MG1655 (24B orf61) 90120 min 3060 min DNA replication have been reported previously [21, 22].
MG1655 (24B orf73) 90120 min 120150 min Suggestions that some genes of 24B prophage may affect
MG1655 (24B ea22) 120150 min 3060 min host growth were also published [38]. However, the results
a
The value was not determined due to a very low efficiency of prophage
described in this subsection demonstrate for the first time
induction under these conditions (as shown in Figure 2). that the exo-xis region can significantly modulate one of
global cellular responses, the S.O.S. response, after treatment
with hydrogen peroxide.
and treated with hydrogen peroxide. In both and 24B , 3.4. Expression of Crucial Phage Genes Is Dramatically
deletion of the exo-xis region caused a significant reduction Decreased after Treatment of Lysogenic Cells with Hydro-
in the mRNA levels of most of the S.O.S. regulon genes gen Peroxide in the Absence of the Exo-Xis Region in
relative to wild-type prophages, with exceptions of rpoS 24B Prophage. Expression of phage genes, crucial for the
E. coli () E. coli (24B )

12 12
10 10
(E-Method)
8 8
(E-Method)
6 6
4 4
2 2
0 0

E. coli (exo-xis) E. coli (24B exo-xis)

12 12

10 10
8
(E-Method)
8
(E-Method)
6 6
4 4
2 2
0 0

Figure 7: Expression of genes from the S.O.S. regulon in E. coli MG1655 lysogenic with , exo-xis, 24B , or 24B exo-xis, at indicated
times after UV irradiation (50 J/m2 ), as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated
cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate
the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with
error bars indicating SD.
regulatory processes and lytic development, has been tested region on expression of a battery of phage genes under
under the same conditions as described in the preceding conditions of the oxidative stress. The results presented in
subsection. The specific conditions and time after addition of Figure 6 for phage are compatible with those published
hydrogen peroxide into the cell culture at which samples were previously (though obtained with different methods) [23], as
withdrawn were chosen on the basis of similar experiments overexpression of the exo-xis region had opposite effects to
reported previously [26]. Interestingly, different effects of the those observed in its absence. On the other hand, severely
deletion of the exo-xis region were observed for phages and impaired expression of all tested phage genes in 24B exo-
24B . In , deletion of genes and open reading frames located xis was unexpected. However, these results (Figure 6) can
between exo and xis genes did not cause considerable effects explain a strong defect in the induction of 24B exo-
on mRNA levels for xis, exo, and Q, whereas expression of int, xis prophage (and perhaps further lytic development) by
cIII, N, cI, cro, cII, O, and R was enhanced upon treatment hydrogen peroxide (Figure 2). Similarly, drastic differences
with hydrogen peroxide (Figure 6). Completely different between effects of exo-xis mutations on hydrogen peroxide-
results were obtained when 24B and 24B exo-xis lysogens mediated prophage induction between and 24B (Figure 2)
were studied; namely, expression of all tested genes was can be ascribed to opposite regulation of expression of phage
drastically impaired in hydrogen peroxide-treated bacteria in genes in the absence of the exo-xis region.
the absence of the exo-xis region on the prophage (Figure 6).
While negative regulation of transcription from cII- 3.5. Effects of the Exo-Xis Region on Expression of Host and
dependent promoters by overexpression of the exo-xis region Phage Gene in UV-Irradiated Lysogenic Cells. Experiments
has been reported previously in phage [23], this study analogous to those described in two preceding subsections
demonstrated for the first time significant effects of this were performed with lysogenic cells irradiated with UV.
12
10
8
6
4
E. coli () E. coli (24B ) 2
60 60
0
int xis exo cIII N cI cro cII O Q R

50 50

40 40
(E-Method)
(E-Method)

30 30

20 20
10
10
0 0
int xis exo cIII N cI cro cII O Q R int xis exo cIII N cI cro cII O Q R
exo- xis + exo- xis +

exo-xis exo-xis
Figure 8: Expression of selected bacteriophage genes in E. coli MG1655 lysogenic with or 24B , either wild-type (blue columns) or exo-
xis (yellow columns) at 160 min after UV irradiation (50 J/m2 ), as estimated by reverse transcription quantitative real-time PCR. The values
obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the
presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments
(biological samples), with error bars indicating SD. The additional panel for E. coli (24B ) represents the results of exo-xis variant with
different scale, due to very small values measured. Statistically significant differences (in t-test) are marked as follows: < 0.05, < 0.01,
and < 0.001.
Interestingly, in both and 24B phages, deletion of the exo- and expression of phage genes crucial for lytic development
xis region caused only moderate effects on expression of (particularly xis, exo, N, cro, O, Q, and R) in 24B , but not
most genes from the S.O.S. regulon (Figure 7), contrary in . Since the oxidative stress appears to be the major cause
to hydrogen peroxide-treated bacteria where the differences of induction of Shiga toxin-converting prophages during
were significantly higher (compare Figures 5 and 7). The infections of human intestine by enterohemorrhagic E. coli
exceptions in UV-irradiated cells were rpoD, rpoH, and rpoS (EHEC), the exo-xis region and/or products of its expression
genes in and rpoH and rpoS in 24B , whose expressions were might be considered as potential targets for anti-EHEC drugs.
at considerably lower level in the absence of the exo-xis region
(Figure 7). One should also note that the induction of the
S.O.S. regulon with UV irradiation was quicker than that with Conflict of Interests
hydrogen peroxide. These results indicate that the influence of The authors declare no conflict of interests.
the exo-xis region on the S.O.S. response is particularly well
pronounced under conditions of the oxidative stress.
Unlike the S.O.S. regulon expression, levels of mRNAs Authors Contribution
of bacteriophage genes in UV-irradiated cells were affected
similarly to those in hydrogen peroxide-treated lysogenic Katarzyna Licznerska and Aleksandra Dydecka contributed
bacteria by the absence of the exo-xis region (Figure 8). Again, equally to this work.
although some differences were observed between and
exo-xis, the differences between 24B and 24B exo-xis Acknowledgment
were dramatic. This indicates that the influence of the exo-xis
region on expression of phage genes after prophage induction This work was supported by the National Science Center
does not depend on the induction agent. (Poland) Grant no. 2013/09/B/NZ2/02366 to Alicja Wgrzyn.
4. Conclusions References
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http://dx.doi.org/10.1155/2016/3863726
Review Article
Modulation of Hypercholesterolemia-Induced
Oxidative/Nitrative Stress in the Heart
Csaba Csonka, Mrta Srkzy, Mrton Pipicz, Lszl Dux, and Tams Csont
Department of Biochemistry, Faculty of Medicine, University of Szeged, Dom ter 9, Szeged 6720, Hungary
Correspondence should be addressed to Tamas Csont; csont.tamas@med.u-szeged.hu
Copyright 2016 Csaba Csonka et al. This is an open access article distributed under the Creative Commons Attribution License,
Hypercholesterolemia is a frequent metabolic disorder associated with increased risk for cardiovascular morbidity and mortality.
In addition to its well-known proatherogenic effect, hypercholesterolemia may exert direct effects on the myocardium resulting
in contractile dysfunction, aggravated ischemia/reperfusion injury, and diminished stress adaptation. Both preclinical and
clinical studies suggested that elevated oxidative and/or nitrative stress plays a key role in cardiac complications induced by
hypercholesterolemia. Therefore, modulation of hypercholesterolemia-induced myocardial oxidative/nitrative stress is a feasible
approach to prevent or treat deleterious cardiac consequences. In this review, we discuss the effects of various pharmaceuticals,
nutraceuticals, some novel potential pharmacological approaches, and physical exercise on hypercholesterolemia-induced
oxidative/nitrative stress and subsequent cardiac dysfunction as well as impaired ischemic stress adaptation of the heart in
hypercholesterolemia.
1. Introduction to cardioprotective interventions including ischemic pre-

and postconditioning [10, 11]. Although the pathoetiology of
Hypercholesterolemia, a frequent form of hyperlipidemia, is hypercholesterolemia has been studied extensively, the pre-
a metabolic disorder characterized by elevated levels of total cise molecular mechanisms leading to cardiac complications
cholesterol in the blood. Hypercholesterolemia may develop are not entirely clear. Nevertheless, substantial evidence exists
as a consequence of unbalanced diet, obesity, inherited demonstrating that hypercholesterolemia induces oxidative
(genetic) diseases (familial hypercholesterolemia), or other and nitrative stress in the heart and that oxidative/nitrative
diseases (e.g., diabetes). According to large clinical stud- stress plays a role in several cardiac pathologies. Therefore,
ies, hypercholesterolemia affects a significant population of modulation of oxidative stress in the hypercholesterolemic
adults in developed countries [1]. For instance, approximately myocardium appears to be a rational approach. In this review
100 million people (44.4%) suffered from hypercholestero- we aim to discuss relevant literature related to potential
lemia (>5.2 mmol/L) in the United States in 2008 [2]. The modulation of hypercholesterolemia-induced oxidative stress
relationship between hypercholesterolemia and cardiovas- and subsequent complications in the heart (Figure 1). Our
cular mortality has been known for decades [3]. Hyper- attention is focused on certain pharmaceuticals, nutraceuti-
cholesterolemia, especially elevated low density lipoprotein cals, novel pharmacological approaches, and physical exercise
(LDL) cholesterol, is a major risk factor for the devel- as potential modulators.
opment of atherosclerosis and subsequent ischemic heart
disease [4], which is a leading cause of death worldwide [5]. 2. Hypercholesterolemia and
Moreover, several experimental studies have demonstrated
that, in addition to its well-known proatherogenic effect
Oxidative/Nitrative Stress
in the vasculature, hypercholesterolemia may directly affect Oxidative/nitrative stress can be defined as an excess for-
the heart causing contractile dysfunction [68], aggravated mation or insufficient removal of highly reactive molecules
ischemia/reperfusion injury [9], and attenuated responses such as reactive oxygen and/or nitrogen species (ROS and
miRNA Pharmaceuticals
Myocardial cell
Nucleus Biological actions

(1) Contractile dysfunction
Gene
(2) Aggravated I/R injury
expression (3) Diminished stress adaptation
Cholesterol Cholesterol
miRNA
Farnesol, geranylgeraniol
Prenylation
Mevalonate NADPH oxidase Antioxidant defense
HMG-CoA reductase (1) Antioxidant enzymes
ONOO , ROS, RNS
O2 (2) Exogenous antioxidants
HMG-CoA Oxidative/nitrative
stress (3) Endogenous antioxidants
Nutraceuticals Ac-CoA NO
Pharmaceuticals
Physical exercise
Statins
Physical exercise Nutraceuticals
eNOS
Figure 1: Hypercholesterolemia-induced myocardial oxidative/nitrative stress and its possible modulations (in grey boxes) to prevent or treat
deleterious cardiac consequences. Ac-CoA: acetyl-coenzyme A; HMG-CoA: 3-hydroxy-3-methyl glutaryl coenzyme A; eNOS: endothelial
nitric oxide synthase; ONOO : peroxynitrite; ROS: reactive oxygen species; RNS: reactive nitrogen species; miRNA: microRNA.
RNS, resp.) including, for instance, superoxide, hydrogen as a direct target of miR-25, suggesting that decreased miR-
peroxide, hydroxyl radical, and peroxynitrite [12]. Enzymatic 25 allows the upregulation of NOX4, which contributes to
sources for ROS formation include the mitochondrial respi- increased ROS production in the hypercholesterolemic heart
ratory chain, nicotinamide adenine dinucleotide phosphate [8]. Although alterations in cardiac gene expression have
(NADPH) oxidases, xanthine oxidase, cyclooxygenases, been demonstrated in animal models of diet-induced hyper-
uncoupled nitric oxide synthase (NOS), and peroxidases, cholesterolemia [14, 15] and metabolic syndrome [16], to the
while antioxidant enzymatic systems include superoxide dis- best of our knowledge, the precise molecular link between
mutase (SOD), catalase, glutathione peroxidase, glutathione hypercholesterolemia and altered cardiac gene expression has
reductase, and heme oxygenase (HO) [13]. Although there not yet been elucidated. Interestingly, two key transcriptional
is a consensus in the literature that hypercholesterolemia is factors have been implicated to date in cholesterol-dependent
associated with increased cardiac oxidative stress (Figure 1), transcriptional regulation of gene expression, that is, the
the precise molecular mechanisms by which hypercholes- sterol-regulatory element binding protein (SREBP) and the
terolemia induces oxidative stress in the heart are not entirely liver X receptor (LXR) [17]; however, direct evidence for their
clear. Accumulating evidence shows increased superoxide role in hypercholesterolemia-induced oxidative stress is still
production in the hearts of hypercholesterolemic animals, lacking.
and one of the major sources of this superoxide appears to be Another possible explanation for increased oxidative
increased NADPH oxidase activity in cholesterol-fed Wistar stress could be diminished endogenous antioxidant capac-
rats and apoB100 transgenic mice [6, 8]. In the hearts of ity. Indeed, hypercholesterolemia has been reported to be
cholesterol-fed CFLP mice and Wistar rats, transcript levels associated with decreased cardiac expression and activity
of NADPH oxidase 4 (NOX4) were significantly increased of antioxidant enzymes [1821]. Nevertheless, the precise
when compared to normal controls fed a standard chow molecular mechanisms by which high cholesterol downreg-
[8, 14]. The increased NOX4 transcript levels in hypercholes- ulates myocardial antioxidant enzymes remain to be elu-
terolemic hearts can be related to transcriptional, posttran- cidated. Besides enzymatic mechanisms, tissue level of the
scriptional, or epigenetic regulation of NOX4 expression. endogenous antioxidants is also an important determinant of
One study shows an association of cardiac NOX4 transcript oxidative/nitrative stress. Nitric oxide (NO) is a key antioxi-
and protein levels as well as NADPH oxidase activity with dant molecule that is synthesized by NO synthases in various
decreased myocardial level of the microRNA miR-25 in cells including cardiomyocytes. In the hearts of hypercholes-
rats with diet-induced hypercholesterolemia [8]. MicroRNAs terolemic animals, cardiac NO level was decreased when
(miR or miRNA) are endogenous, small, noncoding RNAs compared to controls [10, 22, 23]; however, this effect was
that are responsible for posttranscriptional silencing of a independent of the modulatory effect of cholesterol on the
wide variety of specific target genes. NOX4 was identified mevalonate pathway [23]. Moreover, transcript levels of NOS
3 were decreased, while NOS 1 and 2 remained unaffected in The current treatment of myocardial infarction includes the
the hearts of cholesterol-fed mice [14]. attempt to reopen the occluded coronary artery (termed
In addition to the effects of cholesterol and its derivatives reperfusion) in a timely manner by coronary interven-
on gene expression, membrane-related effects of cholesterol tion procedures or thrombolytic therapies in order to
are also plausible [39]; however, these effects have barely been reduce infarct size, which is one of the major deter-
investigated in the hypercholesterolemic heart. minants of long term complications and survival. Thus
the majority of patients with acute myocardial infarc-
3. Functional Effects of tion undergo ischemia/reperfusion injury. However, the
myocardium is remarkably good at adapting to ischemic
Hypercholesterolemia on the Heart conditions by triggering endogenous adaptive mechanisms
Hypercholesterolemia has been shown to exert direct against ischemia/reperfusion injury. One of the most pow-
myocardial effects independent of the development of erful endogenous adaptive cardioprotective mechanisms is
atherosclerosis in both clinical [40, 41] and preclinical ischemic preconditioning, that is, when brief exposure to
studies [7, 8]. These effects include impaired cardiac per- repetitive ischemia/reperfusion cycles markedly enhances the
formance and contractile dysfunction [6, 7], aggravated ability of the heart to withstand a subsequent, potentially
ischemia/reperfusion injury [9], and diminished adaptation lethal ischemic attack [53]. Another possible intervention is
to ischemic stress [10, 11, 42] (Figure 1). termed ischemic postconditioning when the initial phase of
reperfusion is interrupted with short periods of ischemia
3.1. Cardiac Contractile Dysfunction. Observations in human [54]. In addition, remote conditioning has also been intro-
clinical trials and animal models suggest a direct effect of duced when cardioprotection is achieved by exposing an
cholesterol on myocardial contractile function leading to organ at a distance from the heart to ischemia/reperfusion
impaired diastolic and in some cases also systolic function [6 insults [55]. To date, a very large number of preclinical
8, 4345]. Although the symptoms of hypercholesterolemia and clinical studies are available suggesting that hypercho-
are not pronounced initially and the disorder may be dormant lesterolemia enhances the severity of ischemia/reperfusion
for a long time, preclinical diastolic dysfunction shows a clear injury and interferes with the endogenous cardioprotec-
progression to heart failure [46]. Development of heart failure tive mechanisms. The mechanisms of ischemia/reperfusion
aggravates with advancing age and contributes to age-related injury and endogenous cardioprotection as well as the effect
morbidity and mortality [47]. of hypercholesterolemia on these phenomena have been
The direct effects of cholesterol exposure on cardiomy- extensively reviewed elsewhere [5658].
ocyte function were demonstrated in a cell culture model; that
is, elevation of membrane cholesterol content in ventricular
cardiomyocytes resulted in decreased cytosolic calcium levels 4. Modulation of Hypercholesterolemia-
and impaired cardiac myocyte contractility [48]. This has Induced Oxidative/Nitrative Stress
been confirmed in cholesterol-fed rabbits and rats, showing
Modulation of oxidative/nitrative stress in hypercholes-
apparent contractile dysfunction characterized by decreased
terolemia can be approached by at least 3 different ways
maximum rate of shortening, decreased rate of relaxation,
(Figure 1). First, cholesterol-lowering therapies should be
and increased left ventricular end-diastolic pressure [7, 8, 10,
effective in attenuation of oxidative/nitrative stress due to
44]. Moreover, impairment of cardiac performance assessed
their trigger-eliminating effect. Second, the mechanism of
by measuring aortic flow was also demonstrated in hearts
action of several drugs used for cholesterol-lowering is
isolated from hypercholesterolemic apoB100 transgenic mice
complex and may involve antioxidant properties. The second
[6]. Hypercholesterolemia-induced cardiac dysfunction was
approach may be the most obvious, that is, the application
further confirmed by echocardiography in humans [43, 45,
of antioxidant molecules and especially natural products to
49].
reduce oxidative/nitrative stress. Third, support or induction
It has been shown previously that myocardial oxida-
of endogenous enzymatic antioxidant systems or inhibition
tive/nitrative stress induced by hypercholesterolemia signifi-
of the prooxidant enzymes may be a feasible way to control
cantly contributes to the development of cardiac dysfunction
cardiac oxidative/nitrative stress in hypercholesterolemia.
[6, 8, 50]. However, the exact underlying molecular mech-
These distinct mechanisms of action are often combined in
anisms are still not entirely clear. One possible mechanism
the case of certain modulators. In this section we discuss var-
is the oxidation of contractile proteins [8, 51, 52]. In failing
ious pharmaceuticals, nutraceuticals, some novel approaches,
human heart samples, for instance, there is an obvious oxi-
and even physical exercise as potential modulators of
dation and nitrosylation of tropomyosin and actin, showing
hypercholesterolemia-induced oxidative/nitrative stress.
a positive correlation with diminished contractile function
indicated as a decrease in ejection fraction [51]. Moreover,
increased protein oxidation was confirmed in the hearts of 4.1. Pharmaceuticals. To the best of our knowledge, there are
hypercholesterolemic rats [8]. no drugs on the market approved to specifically target cardiac
oxidative/nitrative stress induced by hypercholesterolemia.
3.2. Aggravated Ischemia/Reperfusion Injury and Attenuated Nevertheless, cholesterol-lowering drugs are excessively pre-
Stress Adaptation. Hypercholesterolemia facilitates the risk scribed for patients presenting hypercholesterolemia and
of atherosclerosis and subsequent myocardial infarction. due to their cholesterol-lowering effect they should have
a secondary attenuating effect on hypercholesterolemia- effect of atorvastatin was also shown by several studies [69
induced oxidative/nitrative stress. Interestingly, several of 72]. Moreover, in hyperlipidemic subjects with metabolic
these drugs have also been implicated in directly modulating syndrome, atorvastatin is associated with a greater reduction
oxidative/nitrative stress. Moreover, large clinical trials have in lipid markers of oxidation compared with pravastatin [73].
shown that antihyperlipidemic agents, for example, statins In contrast, Sodha et al. [74] found increased levels of myocar-
[59], fibrates [60], and niacin [61], could reduce the incidence dial biomarkers of oxidative stress in hypercholesterolemic
of cardiovascular events in hypercholesterolemic patients swine treated with atorvastatin. Pitavastatin, the newest
[59]. In addition, some drugs used for other indications than member of the HMG-CoA reductase inhibitor family, has
treatment of hypercholesterolemia (e.g., some antidiabetics shown improvements in both cardiovascular function and
and vasodilators) have also been demonstrated to attenuate markers of oxidative stress [75], presumably via decreasing
hypercholesterolemia-induced oxidative/nitrative stress and NADPH oxidase activation [64]. Fluvastatin was also shown
deleterious cardiac consequences. to play a protective role against high-cholesterol-induced
oxidative stress and DNA damage [76]. Taken together, the
4.1.1. Statins. Statins are a class of cholesterol-lowering drugs cardioprotective effect of almost every statin is associated
that inhibit the key enzyme, 3-hydroxy-3-methylglutaryl with some pleiotropic effect which can decrease oxidative
coenzyme A (HMG-CoA) reductase, of the endogenous stress in the cardiovascular system.
cholesterol biosynthesis. Statins have therefore become estab-
lished in the treatment of hypercholesterolemia and attained 4.1.2. Ezetimibe. Ezetimibe, another lipid-lowering drug, is
a central place in cardiovascular medicine [62]. However, used in monotherapy or in combination with statins and
there are potential side effects of statin therapy including is responsible for lowering intestinal cholesterol absorption
skeletal muscle complaints and/or mild elevation of serum by inhibiting the Niemann-Pick C1-Like 1 (NPC1L1) sterol
creatine kinase level and very rarely rhabdomyolysis [63]. transporter. Clinical studies showed that ezetimibe attenuates
The main members of statins on the market are lovastatin, the markers of oxidative stress in hyperlipidemic subjects
rosuvastatin, simvastatin, atorvastatin, fluvastatin, pravas- [7780]. The drug is suggested to exert both cholesterol-
tatin, and the newest addition, pitavastatin. By inhibition dependent and independent actions [7880]. Reduction of
of the formation of mevalonate, the direct reaction product serum cholesterol results in decreased cholesterol influx
of HMG-CoA reductase and HMG-CoA, statins not only to the cells thereby attenuating cholesterol-induced oxida-
inhibit the formation of the end-product, cholesterol, but tive stress. For instance, ezetimibe reduced hepatic choles-
also they reduce the formation of other cholesterol pathway terol level in obese male mice which in turn attenuated
intermediers such as the 15-carbon isoprenoid farnesol or the oxidative stress via downregulation of NADPH oxidases,
20-carbon isoprenoid geranylgeraniol which finally lead to cytochrome P4502E1, and beta oxidation [81]. In addition
reductions in protein prenylation, Coenzyme Q10 (see later), to its cholesterol-lowering action, ezetimibe may exert a
and dolichol synthesis. Besides lipid-lowering effects, statins direct cholesterol-independent effect on cells [7880, 82].
have pleiotropic effects on different cell types. Some of these A feasible explanation is that NPC1L1 is widely expressed
cholesterol-independent effects of statins involve improved in many other tissues including liver, kidney, muscle, and
endothelial function, stabilization of atherosclerotic plaques, heart [83]. Cell culture studies support the possible direct
and attenuation of oxidative stress and inflammation as effect of ezetimibe on cellular oxidative/nitrative stress, for
well as inhibition of the thrombogenic response [64, 65]. example, knockdown of NPC1L1 in hepatocytes attenuated
Pleiotropic effects may play an important part in reducing ROS production [84]. Based on these findings it is plausible
cardiovascular mortality and morbidity and may act at to speculate that ezetimibe has a direct impact on the
multiple points in the complex cascade of events leading to heart and may decrease cholesterol-induced oxidative stress.
atherosclerosis. Nevertheless, direct cardioprotective effect of ezetimibe has
Regarding the antioxidative effect of statins, a recent not yet been investigated in the literature; thus preclinical
study showed that, beside the cholesterol-lowering effect, studies needed to address this issue.
simvastatin is able to ameliorate endothelial dysfunction
through increasing NO bioavailability and through suppres- 4.1.3. Niacin. Although niacin (nicotinic acid or Vit B3 ) is
sion of oxidative stress in a rat model of hypercholesterolemia a potent lipid-lowering agent when used in pharmacologic
[66]. Similar results were shown by Iliodromitis et al., doses, its clinical use is limited by side effects, so it can
[67] who found that a 3-week simvastatin treatment limits be applied particularly in combination to treat marked
infarct size and attenuates oxidative and nitrosative stress dyslipidemia with strict monitoring ([85], for review see
both in normocholesterolemic and in hypercholesterolemic [86]). Niacin administration was shown to reduce markers of
rabbits subjected to ischemia/reperfusion irrespective of the serum oxidative stress and increase antioxidant paraoxonase-
presence of postconditioning, while postconditioning was 1 in patients with hypercholesterolemia [87]. A randomized
effective only in normocholesterolemic animals. According controlled clinical trial reported that niacin improves both 6-
to another study [68], pravastatin, in contrast to same- and 15-year mortality of patients after myocardial infarction
dose simvastatin or postconditioning, was found to reduce with or without metabolic syndrome thereby suggesting a
infarction in hypercholesterolemic rabbits independently of cardioprotective effect for niacin [88]. Direct cardioprotective
lipid-lowering, potentially through eNOS activation and effect of niacin was reported in preclinical studies as well
attenuation of oxidative/nitrative stress. The antioxidative [8992]. However, evidence is not available to clarify the
potential beneficial effect of niacin in cholesterol-induced Increasing the level of glucagon-like peptide-1 by admin-
cardiac stress. istration of analogues or by inhibiting dipeptidyl peptidases is
used for the treatment of diabetes and has recently emerged
4.1.4. Fibrates. Fibrates (amphipathic carboxylic acid deriva- as a potential cardioprotective approach [111]. Specifically, a
tives) lower plasma triglyceride and small LDL level and glucagon-like peptide-1 agonist was shown to reverse cardiac
increase high density lipoprotein (HDL) concentration; thus dysfunction induced by high-fat diet [112] and a dipeptidyl
fibrates are particularly used to treat combined dyslipidemia peptidase inhibitor was found to improve cardiac dysfunction
related to diabetes or metabolic syndrome [93]. This class and oxidative stress in high-fat high-fructose-fed mice [113].
of drugs acts on nuclear peroxisome proliferator-activated The vasodilator fasudil (a potent rho kinase inhibitor)
receptor (PPAR), leading to transcriptional changes [94]. was shown to lower serum cholesterol level and decrease
Studies revealing the effect of fibrates on cholesterol-induced cardiac oxidative stress by enhancing antioxidants in hyperc-
cardiac oxidative stress are not available in the literature. Nev- holesterolemic rat hearts [114]. The group has reported that
ertheless, some investigations have reported an antioxidant fasudil restores cardiac eNOS and NO level diminished by
effect of fibrates. A subcohort clinical trial has demonstrated high-cholesterol diet [114]. Interestingly, even in the state of
that 3-week fenofibrate treatment reduced ox-LDL and 8- hypercholesterolemia, fasudil can induce both precondition-
isoprostane, systemic markers of oxidative stress in patients ing and postconditioning [115]. Moreover, fasudil restores the
with hypertriglyceridemia [95]. In accordance with these cardioprotective effect of ischemic postconditioning in rats
results, fenofibrate decreased ox-LDL with a modest reduc- with hypercholesterolemia [116].
tion in cholesterol level in healthy normolipidemic older
adults [96]. Fibrates may exert antioxidant actions indepen- 4.2. Nutraceuticals. In spite of the extensive research in
dently from their lipid-lowering effect. For instance, Sugga et cardiology, particularly in ischemic heart diseases, only
al. have reported that in vivo administration of fenofibrate and very few new cardioprotective drugs have found their way
clofibrate before ex vivo myocardial ischemia/reperfusion into clinical practice [117]. Therefore, there is a growing
reduced infarct size and oxidative stress [97]. Others have interest in nutraceuticals to treat and also prevent certain
shown antioxidant and cardioprotective effects of fibrates cardiac diseases or restore the injured adaptation of the
against development of ventricular hypertrophy associated heart. Nutraceuticals is a term that refers to a wide range
with oxidative stress [98, 99]. Furthermore, fibrates prevent of products including but not limited to natural herbal
endothelial dysfunction by ameliorating oxidative stress, products, dietary supplements, isolated nutrients, and special
without affecting plasma lipid levels [100, 101]. It has been diets. Since there is also a growing desire among potential
also published that knockout of PPAR, the ligand of fibrates, customers to consume natural dietary products instead of
leads to oxidative stress associated with cardiac dysfunction chemically synthesized compounds, the importance of these
[102]. In contrast, fenofibrate failed to improve lipotoxic nutraceuticals is being further emphasized.
cardiomyopathy [103], and high dose fibrate induced cardiac Natural food substances have the potential to alter bio-
damage in healthy rats [104]. Taken together, the studies logical functions of the cells by mechanisms enhancing the
focusing on antioxidant action of fibrates suggest a potential endogenous antioxidant systems or through altering the
beneficial effect on cholesterol-induced cardiac oxidative redox signaling status of the cell. This is often related to the
stress, although this needs to be confirmed in further studies. unique composition of different antioxidant compounds in
the various nutraceuticals. These products could be beneficial
4.1.5. Other Pharmaceuticals. There are some available drugs, in pathological conditions where oxidative stress plays an
which are basically used to treat other diseases than hyper- important role.
cholesterolemia and which have beneficial effects on high-
cholesterol-induced cardiac consequences. 4.2.1. Antioxidant Vitamins. A number of preclinical stud-
Rosiglitazone is an antidiabetic drug; however, it also ies have demonstrated that classical antioxidant vitamins
improves blood cholesterol level in hypercholesterolemic including beta carotene (Vit A precursor), folic acid (Vit
models [105, 106]. It was reported that the drug pre- B9 ) vitamin C, and vitamin E administered alone or in
vents the development of high-cholesterol-induced cardiac combination with other drugs or in multivitamin prepa-
hypertrophy [105] and ameliorates postischemic recovery rations could improve serum lipid profile and myocardial
and nitrative stress in the heart of high-cholesterol-fed oxidative/nitrative stress in hypercholesterolemia [18, 118
rabbits [107]. The latter research group has also shown 126]. Moreover, low dose beta carotene has been shown to
that rosiglitazone alleviates aggravated postischemic myocar- improve cardiac function and reduce myocardial oxidative
dial injury and myeloperoxidase upregulation caused by stress after ischemia/reperfusion injury in hearts of rats [63].
hypercholesterolemia, independently from the lipid-lowering In contrast, vitamin C alone or combined with vitamin E
action [108]. Rosiglitazone has a direct antioxidant effect failed to reduce myocardial infarct size in an open chest rabbit
on cardiomyocytes [109] and has been demonstrated to model [127]. Indeed, Trolox, a vitamin E analogue did not
prevent the upregulation of cardiac NOX4 in a high-fat high- reduce infarct size but accelerated functional recovery after
sugar diet, streptozotocin-induced diabetic model associated myocardial infarction in porcine hearts [128]. In addition,
with dyslipidemia [110]. These findings make rosiglitazone a we have previously shown that a multivitamin preparation
promising modulator of cholesterol-induced cardiac oxida- supplemented with phytosterols decreased serum choles-
tive stress. terol level in hypercholesterolemic rats; however, it did
not reduce infarct size either in normocholesterolemic or oxidation and consequently the progression of atheroscle-
in hypercholesterolemic hearts of rats [118]. In contrast, rosis. It also decreased ischemia/reperfusion injury after
preconditioning with vitamin E has been shown to improve myocardial infarction [135, 136]. Significant improvement has
postischemic contractile and vascular functions in hearts of been demonstrated in clinical and hemodynamic parameters
rats after ischemia/reperfusion injury [129]. To the best of and in exercise tolerance in patients given adjunctive CoQ10
our knowledge, the effects of antioxidant vitamins have not in various trials conducted in patients suffering from heart
been tested on cardioprotection conferred by ischemic pre- failure, hypertension, ischemic heart disease, and other car-
or postconditioning. However, decrease of oxidative/nitrative diac illnesses [135137]. Therefore, CoQ10 could be a potential
stress by antioxidant vitamins could be a potential therapeutic therapeutic molecule in heart diseases.
target in the restoration of cardioprotective adaptive mecha- Due to the common biosynthetic pathway of cholesterol
nisms lost in hypercholesterolemia. and CoQ10, the HMG-CoA reductase inhibitor statins may
Despite a number of preclinical studies proving beneficial
potentially reduce the levels of CoQ10 in different tissues
effects of antioxidant vitamins on cardiac pathologies in
[138]. Indeed, it has been reported that CoQ10 levels in the
hypercholesterolemia, clinical trials investigating the effects
plasma, platelets, and lymphocytes were decreased after statin
of antioxidant vitamins on cardiovascular morbidity and
treatment [138]. Some papers indicated that CoQ10 depletion
mortality have been disappointing. The complex reasons that
might explain the translational failures of preclinical results during statin therapy might be associated with subclinical
into clinical therapy are discussed in detail in recent reviews cardiomyopathy and this situation is reversed upon CoQ10
[130132] and include the following: (i) oxidative/nitrative treatment [138]. In contrast, a preclinical study reported
stress is just a late consequence of cardiac pathologies and that coadministration of CoQ10 with simvastatin impaired
not the primary cause; (ii) pathogenesis of cardiovascular mitophagy and cardioprotection after ischemia/reperfusion
diseases is complex and increased oxidative/nitrative stress is injury in mice and cardiomyocytes [139]. These results raise
not the only cause of these disorders; (iii) antioxidant vitamin the concern that CoQ may interfere with the anti-ischemic
therapy is not able to reduce oxidative/nitrative stress due to benefit of statins mediated through stimulation of mitophagy.
inappropriate study design (inappropriate patient selection, Therefore, patients treated with statins should be also mon-
failure in the administration route, suboptimal time and itored for their CoQ10 status and clinicians should be aware
duration of antioxidant therapy, poor target specificity, and of the aforementioned interaction between statins and CoQ10
potential interaction with other drugs, etc.); (iv) a single as well as the ability of statins to impair skeletal muscle and
antioxidant therapy is not enough to overcome increased myocardial bioenergetics [138].
oxidative/nitrative stress; (v) antioxidant molecules might
have harmful effects on physiological processes or com- 4.2.3. Flavonoids. Flavonoids are a large family of natural
pensatory mechanisms in pathologic conditions induced by polyphenolic compounds in the human diet and their benefi-
oxidative/nitrative stress. cial effect on cardiovascular diseases is widely studied. These
The lack of beneficial effects of antioxidant vitamins compounds favorably affect a wide range of biological pro-
on cardiovascular pathologies in clinical trials does not cesses. Beside their antioxidant capacity, flavonoids improve
disprove that oxidative/nitrative stress plays a crucial role in lipid profile and have anti-inflammatory, antiplatelet, and
cardiac pathologies in hypercholesterolemia. These clinical antithrombotic effects as well.
trials challenge us to develop better antioxidant approaches
and better preclinical models as well as to design more Green Tea Catechins (GTC). Catechins are abundant polyphe-
appropriate clinical trials considering the aforementioned nols in green tea. GTC were shown to reduce blood choles-
reasons of translational failure. terol level in both animals [140143] and humans [144
147]. In addition, GTC reduced accumulation of cholesterol
4.2.2. Coenzyme Q. Coenzyme Q10 (CoQ10), also called
in the rat myocardium in a state of diabetic dyslipidemia
ubiquinone or ubiquinol, is an isoprene derivative and an
[141]. In a double-blind, placebo-controlled clinical study,
alternative product of the mevalonate/cholesterol pathway.
CoQ10 is an endogenous nonenzymatic lipophilic antioxi- obese, hypertensive patients had decreased LDL-cholesterol
dant and free radical scavenging molecule playing a role in the and increased HDL and total serum antioxidant levels after 3
mitochondrial electron transport in the inner mitochondrial months of GTC supplementation [148]. Mehra et al. demon-
membrane [133]. Moreover, CoQ10 has been reported to play strated that catechin hydrate improved high sucrose, high-fat-
part in many levels of the redox control of cellular signaling; induced cardiac lipid peroxidation and activity of antioxidant
in fact the autoxidation of its semiquinone form, generated enzymes [149]. GTC were reported to beneficially affect high-
in various membranes during electron transport, can be a fructose diet-associated hypercholesterolemia and cardiac
primary source for hydrogen peroxide production, which signaling pathways related to lipid metabolism and inflam-
activates transcription factors [134]. Due to its localization in mation [150]. Age-related cardiac oxidative stress was also
the mitochondria, it plays a key role in the cellular bioenerget- improved by GTC [151]. The effect of GTC on hypercholes-
ics especially in tissues with high energy requirements such terolemia associated with cardiac dysfunction is not known,
as the myocardium which is extremely sensitive to CoQ10 but it was reported that GTC ameliorates myocardial function
deficiency [133, 135]. CoQ10 has been shown to decrease after ischemia [152, 153] in pressure-induced chronic heart
the levels of proinflammatory cytokines and to reduce LDL failure [154] and in autoimmune myocarditis as well [155].
Troxerutin. Troxerutin can be isolated from the Japanese decreased total and LDL cholesterol and attenuated oxidative
pagoda tree (Sophora japonica). To the best of our knowledge, stress in the plasma [176178]. The effect of silymarin on
the antioxidant effect of troxerutin on the heart was first hypercholesterolemia-induced myocardial oxidative stress
described in an ischemia/reperfusion model [156]. Geetha is not yet investigated, but some studies indicate that
et al. have investigated the cardiac impact of troxerutin in silymarin exerts cardioprotection against high-cholesterol-
high-fat high-fructose mice model. They have found that mediated oxidative stress. Silymarin attenuates myocardial
troxerutin reduces cholesterol content and oxidative stress ischemia/reperfusion injury by modulating oxidative stress
markers in both the plasma and the heart and increases [179] and silibinin has a direct antioxidant effect on H9c2
cardiac enzymatic and nonenzymatic antioxidant levels [157]. cardiac cells against oxidative stress [180]. Moreover, silibinin
Moreover, the same group have shown in the same dyslipi- was reported to improve both plasma and cardiac cholesterol
demic model that troxerutin reverses fibrotic changes in the content and attenuate oxidative markers and degenerative
heart and improves cardiac function probably by reducing changes in the heart of animals exposed to arsenic [181].
ROS production [158]. In this study, silibinin prevented cardiac oxidative stress by
inhibiting the induction of prooxidants (e.g., NOX2 and
Quercetin. The cardioprotective effect of the dietary flavonoid NOX4) and enhancing antioxidants.
quercetin (found in many fruits, vegetables, leaves, and
grains) against ischemia/reperfusion injury is well studied Naringin and Hesperidin. Naringin and hesperidin are nat-
[159163]; however, few studies are available in the litera- ural flavonglycosides in citrus fruits. Many studies reported
ture regarding the impact on hypercholesterolemia-induced hypocholesterolemic effect of naringin and hesperidin in ani-
oxidative stress in the heart. It was reported that quercetin mal models [182188] and in a clinical study [189]. However,
ameliorates left ventricular function, collagen deposition, and in moderately hypercholesterolemic subjects, naringin and
inflammatory cell infiltration in rats with metabolic syn- hesperidin failed to lower serum cholesterol [190]. Alam
drome [164]. In that study, quercetin increased the expression et al. have published that naringin improves ventricular
of the transcription factor Nrf2 and the enzyme heme oxy- diastolic dysfunction, cardiac inflammatory cell infiltration,
genase as protective proteins against oxidative stress. Inter-
and plasma cholesterol in high carbohydrate, high-fat-fed
estingly, quercetin exerted a cardioprotective effect without
rats [182]. Recently, naringin has been shown to protect
reducing plasma cholesterol level thereby suggesting a direct
against hypercholesterolemia-induced oxidative stress in the
cardiac effect. In other studies, quercetin has been shown
heart [191]. The study has demonstrated that naringin amelio-
to reduce serum LDL and increase HDL cholesterol level in
hypercholesterolemic rats [165] and lower total cholesterol rates cardiac lipid accumulation and cardiac oxidative stress
in rabbits fed a high-cholesterol [166] and high-fat [167] markers by enhancing enzymatic and nonenzymatic antiox-
diet. Ulasova et al. showed that quercetin improves lipid idants. Moreover, tissue and serum markers of cholesterol-
profile of ApoE knockout mice and prevents ventricular induced cardiac damage were also attenuated by naringin
hypertrophy without affecting myocardial function [168]. In supplementation [191]. Although hesperidin was reported to
a cadmium-induced toxicity study, quercetin was found to protect against cardiac injury induced by doxorubicin [192]
reduce cardiotoxicity and dyslipidemia by attenuating cardiac or ischemia [193, 194], its role in cholesterol-induced cardiac
oxidative stress and lipid parameters [169]. oxidative stress is not known.
Rutin. Rutin (the glycoside between the quercetin and the 4.2.4. Resveratrol. Pleiotropic beneficial effects of the poly-
disaccharide rutinose) improves cholesterol level in different phenol resveratrol (food sources include the skin of grapes,
hypercholesterolemic [170, 171] and dyslipidemic [172, 173] blueberries, raspberries, and mulberries) have been exten-
rodent models. Panchal et al. have shown that rutin supple- sively studied and many publications indicate its protective
mentation ameliorates blood cholesterol level, cardiac struc- function on cholesterol-induced cardiac oxidative stress. A
ture, function, inflammation, and oxidative stress related direct antioxidant effect of resveratrol was demonstrated
to high carbohydrate high-fat-induced dyslipidemia [173]. on H9c2 cardiac cells [195]. Louis et al. have demonstrated
In a streptozotocin-induced diabetes model associated with that resveratrol ameliorates cardiac relaxation dysfunction
hyperlipidemia, rutin attenuated serum cholesterol level and in high-fat-fed rats with hypercholesterolemia [196]. They
myocardial necrosis and improved left ventricular dysfunc- have also shown reduced oxidative stress and inflammatory
tion [174]. It was also reported that rutin exerts cardioprotec- markers in the serum as a result of resveratrol treatment [196].
tion by attenuating oxidative stress and dyslipidemia induced In hypercholesterolemic rats with normal cardiac function,
by high fluoride administration in rats [172]. resveratrol improved postischemic recovery of the heart
[197]. Large animal studies from the same research group
Silymarin. Silymarin is a mixture of three flavonolignans reported that resveratrol improved high-cholesterol and
(silibinin, silydianin, and silychristin) extracted from milk chronic ischemia-induced cardiac dysfunction and oxidative
thistle seeds (Silybum marianum). Krecman et al. demon- damage of myocardial proteins [198200]. The group also
strated that silymarin prevented the development of hyper- described that resveratrol improved high-cholesterol-
cholesterolemia in high-cholesterol-fed rats [175]. They have induced myocardial dysfunction without decreasing protein
also shown that silibinin itself was not as effective as sily- oxidation in the absence of ischemia [198]. Chu et al. have
marin. Others also reported that silymarin or its fraction shown that red wine ameliorates cardiac dysfunction and
oxidative stress in hypercholesterolemic swine subjected 4.2.8. Red Palm Oil. Red palm oil (RPO) is a product from the
to chronic ischemia [50]. Resveratrol was reported to fruits of the oil palm tree (Elaeis guineensis). RPO depending
improve lipid profile and cardiac dysfunction related to on the producer consists of about 51% saturated fatty acids
hyperlipidemia in streptozotocin-induced diabetes [201]. (SFAs), 38% monounsaturated fatty acids (MUFAs), 11%
Finally, these results may promote new studies focusing on polyunsaturated fatty acids (PUFAs), and a spectrum of
cholesterol-induced cardiac oxidative stress. antioxidative carotenoids with tocopherols and tocotrienols
as the major constituents [212]. Other minor components
4.2.5. Grape Seed. Grape seed and skin are rich in natural present in this oil are ubiquinones (mainly CoQ10) and phy-
antioxidants, so these are possible supplements to alleviate tosterols. Red palm oil is therefore a natural carotenoid rich
hypercholesterolemia-induced oxidative stress. In a high- oil that has the potential to act as a very potent antioxidant
fat-induced obesity model, grape seed extract was shown [213]. Red palm oil contains the highest concentration of
to improve lipid profile and prevent postischemic heart tocotrienols compared with other vegetables or plants and
dysfunction and cardiac lipid accumulation along with atten- Serbinova et al. showed that tocotrienols can be 4060 times
uated oxidative stress [202, 203]. Lee et al. reported that more potent as antioxidants than tocopherols [214].
grape skin ameliorates total serum antioxidant capacity of Van Rooyens research group investigated the effect of
rats with high-fat diet and low-fat diet [204]. Antioxidant dietary administration of RPO on the heart. They showed
effect of grape seed was tested on cardiomyoblast H9c2 cell that prolonged dietary feeding with RPO-supplemented diet
culture [205], where it increased endogenous antioxidant (7 g RPO/kg diet) protected the heart against ischemia/re-
systems and prevented ROS-induced apoptosis [205]. In vivo perfusion injury in rats. They used isolated heart mod-
pretreatment with grape seed proanthocyanidins improved els, perfused both in Langendorff technique [215218] and
postischemic functional recovery and reduced ROS pro- working mode [212, 219], showing the vascular-independent
duction in normocholesterolemic rats [206] and partially direct cardioprotective effects of RPO. RPO administration
restored the harmful cardiac effects of hypercholesterolemia improved cardiac function during reperfusion [212] and
via their ability to reduce ROS in the myocardium [207]. decreased infarct size [215, 216]. The real advantage of RPO
administration became apparent when RPO was given to
hyperlipidemic rats. Hyperlipidemia was induced by feeding
4.2.6. Sour Cherry Seed Extract. Based on the observation animals with 2% cholesterol-enriched diet for 59 weeks.
that cherries contain bioactive phytochemicals, for example, This model was characterized by mild cholesterol elevation
phenolics and anthocyanins, which are reported to pos- but a marked decrease of cardiac performance. RPO was
sess antioxidant, anti-inflammatory, anticancer, antidiabetic, able to markedly increase aortic output recovery after 25 min
and antiobesity properties, Tosaki and his group hypothe- global ischemia [219, 220], or infarct size [215] after 30 min
sized that the seed kernel of sour cherry (Prunus cerasus) global ischemia, showing the protective effect of RPO in the
may contain different bioactive constituents [208]. They presence of comorbidities.
demonstrated that kernel extract obtained from sour cherry The proposed mechanism by which RPO exerts its
seed improves postischemic cardiac functional recovery and cardioprotective effect in animals fed high-cholesterol diet
the incidence of ventricular fibrillation and tachycardia in is not fully understood. Supplementation with RPO in the
isolated working rat hearts [209]. Moreover, sour cherry presence of potentially harmful cholesterol showed no sig-
seed extract-induced improvement in cardiac function after nificant difference in serum cholesterol level; therefore, its
ischemia/reperfusion along with decreased atherosclerotic protection cannot be explained by the cholesterol-lowering
plaque formation and infarct size was also observed in hyper- effect of RPO. It is proposed that the protective effect
cholesterolemic New Zealand rabbits fed a 2% cholesterol- of RPO in high-cholesterol diet may be associated with
enriched diet for 16 weeks [210]. In this model, the authors either the RPO antioxidant characteristics and/or changes
demonstrated an increased HO-1 and cytochrome c oxi- in the fatty acid composition of the myocardium during
dase III protein expression following administration of sour ischemia/reperfusion.
cherry seed extract as a possible mechanism of action [210,
211]. 4.3. Promising Novel Approaches to Modulate
Hypercholesterolemia-Induced Oxidative Stress
4.2.7. Spices. Aqueous extracts of certain spices including 4.3.1. miR Modulation. Only 3% of the human genome codes
garlic (Allium sativum), ginger (Zingiber officinale), and for proteins and the remaining part consist of noncoding
cayenne pepper (Capsicum frutescens) as well as their mix- RNAs including short microRNAs (miRNAs, miRs; approx-
ture were shown to attenuate cardiac lipid peroxidation imately 1825 nucleotides in length) [221, 222]. miRNAs can
induced by high-cholesterol, high-fat diet in a rat model inhibit the translation or promote mRNA degradation by
of hypercholesterolemia [20]. Moreover, in the same study, binding to specific mRNAs according to the complementarity
hypercholesterolemia-induced decrease in the myocardial of their seed sequences [223]. Individual miRNAs may simul-
activities of antioxidant enzymes (i.e., SOD, glutathione taneously target multiple mRNAs. However, the expression
peroxidase, and glutathione reductase) was also markedly of individual mRNAs can be regulated by multiple miRNAs.
attenuated by administration of the individual spices as well Therefore, miRNAs may act as fine tuners or as on/off switch-
as their combination [20]. ers of gene expression [223, 224]. Dysregulation of miRNAs
Table 1: Regulation of miRNAs in hyperlipidemia.
Regulation of Regulation of
miRNA Organ Target Role References
miRNA target
let-7g down Aorta ox-LDL receptor 1 up ox-LDL cholesterol uptake [24]
miR-25 down Heart NOX4 up Oxidative stress [8]
Liver, Reverse cholesterol
miR-33 up ABCA1 down [25]
macrophages transport
Peripheral Reverse cholesterol
miR-33 up ABCG1 down [25, 26]
tissues transport
Fatty acid
miR-33 down Liver down VLDL synthesis [27]
synthesis
Fatty acid
miR-33 down Liver up VLDL synthesis [27]
oxidation
Reverse cholesterol
miR-144 up Liver ABCA1 down [28]
transport
HMG-CoA
miR-223 up Liver down Cholesterol biosynthesis [29]
synthase 1
Scavenger
miR-223 up Liver down HDL cholesterol uptake [29]
receptor B1
miR-208a up Heart MED13 down Glucose tolerance [30]
miR-378 and
up Liver MED13 down Glucose tolerance [31]
miR-378
Insulin
miR-378 and Fatty acid
up dependent down Obesity [31]
miR-378 oxidation
tissues
ABC: ATP-binding cassette transporter; HDL: high density lipoprotein; VLDL: very low density lipoprotein; MED: mediator complex subunit; NOX: NADPH
oxidase.
in pathological conditions may alter gene networks; therefore has been demonstrated in cardiac hypertrophy induced by
miRNA replacement or antisense inhibition therapy offers transverse aortic constriction surgery in mice; however, there
a new approach to treating diseases by modulating gene is no data published on increased oxidative/nitrative stress
pathways rather than single molecular targets [222]. or cholesterol levels in that study [237]. Furthermore, in vivo
In recent years, a growing body of evidence has demon- inhibition of miR-25 by a specific antagomir resulted in the
strated that miRNAs play a role in the development of spontaneous development of cardiac dysfunction and sensi-
numerous cardiovascular diseases. Several excellent reviews tized the myocardium to develop heart failure in a Hand2-
focus on miRNAs as diagnostic markers and potential ther- dependent manner [237]. In contrast, inhibition of overex-
apeutic targets in cardiac pathologies including acute coro- pressed miR-25 was reported to ameliorate contractile dys-
nary syndrome [221, 225] and remodelling after myocardial function by improving sarco/endoplasmic reticulum Ca2+ -
infarction as well as heart failure [25, 221, 224, 226228] ATPase (SERCA)2a activity and Ca2+ handling in chronic
and their risk factors including hypercholesterolemia [229], heart failure induced by transverse aortic constriction surgery
diabetes mellitus [230, 231], arterial hypertension [232, 233], in mice as well as in failing human heart samples; however,
atherosclerosis [234, 235], and aging [227]. data on the presence of hypercholesterolemia in humans were
Hypercholesterolemia is a well-known risk factor of lacking in this study [238].
cardiovascular diseases and it leads to increased oxida- It is well known that hyperlipidemia, obesity, metabolic
tive/nitrative stress in the myocardium. Experimental data syndrome, and heart failure are associated with abnormal
are very limited on the regulatory role of miRNAs in cardiac metabolism. In obese mice, a heart specific miRNA,
hypercholesterolemia-induced oxidative/nitrative stress in miR-208a, has been reported to negatively regulate mediator
cardiac pathologies. We have previously shown that the complex subunit 13 (MED13), which controls transcription
myocardial downregulation of miR-25 results in the upreg- by thyroid hormone and other nuclear hormone receptors
ulation of NADPH oxidase 4 (NOX4) mediating oxida- [30] (Table 1). Indeed, cardiac-specific overexpression of
tive/nitrative stress and subsequent myocardial dysfunction MED13 or pharmacologic inhibition of miR-208a in mice
in male hypercholesterolemic rats [8] (Table 1). Moreover, has been demonstrated to confer resistance to high-fat diet-
in a recent study, decreased circulating miRNA-25 level has induced obesity and improve systemic glucose tolerance
been related to the level of oxidative stress indicators in septic [30] (Table 1). Moreover, mice genetically lacking miR-378
patients and the clinical accuracy of miRNA-25 for sepsis and miR-378 have been shown to be resistant to high-fat
diagnosis has been reported to be better than C-reactive pro- diet-induced obesity and exhibit enhanced mitochondrial
tein [236]. In addition, downregulation of miR-25 expression fatty acid metabolism and elevated oxidative capacity of
insulin-target tissues [31] (Table 1). Interestingly, MED13 Table 2: miRNAs affected by ischemic pre- or postconditioning in
and carnitine O-acetyltransferase, a mitochondrial enzyme the heart.
involved in fatty acid metabolism, are among the many targets I/R versus Ipost versus
of miR-378 and miR-378 [31] (Table 1). Thus, these miRNAs miRNA
control
Ipre versus I/R
I/R
provide potential therapeutic targets in hyperlipidemia and
let-7b down [32] n.a. [32] up [32]
metabolic disorders, although their myocardial function
n.a. [35],
needs to be further investigated. miR-21 down [33] up [34, 35]
up [36]
High level of LDL cholesterol and low level of HDL
miR-125b down [32] up [32] up [32]
cholesterol are both well known as independent risk factors
of coronary artery diseases. miRNAs have been shown to reg- miR-139-3p down [32] up [32] up [32]
ulate lipoprotein metabolism and their pro-/antiatherogenic miR-181a down [32] down [32] up [32]
effects at many levels in different tissues as reviewed by others miR-199a down [33] up [34] no data
[25, 222, 229]. Cholesterol efflux from cells is the first step miR-328 down [32] n.a. [32] up [32]
in reverse cholesterol transport to the liver carried out by miR-335 down [32] n.a. [32] up [32]
HDL. miR-33 has been reported to modulate cholesterol
miR-503 down [32] n.a. [32] up [32]
efflux by repressing the expression of ATP-binding cassette
transporter (ABC) A1 in the liver and ABCA1 as well as let-7e n.a. [32] n.a. [32] up [32]
ABCG1 in peripheral tissues [25] (Table 1). Moreover, anti- let-7i n.a. [32] n.a. [32] up [32]
miR-33 therapy was shown to induce the expression of n.a. [32], up [32, 37],
miR-1 up [34]
ABCA1 in macrophages in atherosclerotic plaques thereby down [37, 38] down [35]
reducing the plaque size and local inflammation in mice miR-139-5p n.a. [32] up [32] n.a. [32]
[26] (Table 1). In addition, anti-miR-33 therapy in nonhuman miR-188 n.a. [32] up [32] up [32]
primates increased the expression of miR-33 target genes miR-192 n.a. [32] up [32] n.a. [32]
involved in fatty acid oxidation and reduced the expression of
miR-212 n.a. [32] up [32] n.a. [32]
genes associated with fatty acid synthesis in the liver resulting
in a marked suppression of plasma VLDL triglyceride levels miR-532 n.a. [32] up [32] up [32]
[27] (Table 1). Another miRNA, miR-144, has been reported up [34],
miR-133a no data up [37]
to decrease hepatic ABCA1 expression and plasma HDL down [37]
cholesterol levels [28] (Table 1). Moreover, miR-223 has been miR-208a up [32, 34] n.a. [32] down [32]
demonstrated to reduce HDL cholesterol uptake by decreas- up [34],
miR-320 down [32] down [32]
ing the expression of scavenger receptor B1 and to decrease down [32]
cholesterol biosynthesis through the direct repression of miR-487b up [32] down [32] n.a. [32]
HMG-CoA synthase 1 in the liver of ApoE/ mice [29] I/R: ischemia/reperfusion; Ipre: ischemic preconditioning; Ipost: ischemic
postconditioning; n.a.: not affected.
(Table 1). Furthermore, genetic ablation of miR-223 resulted
in elevated hepatic and plasma total cholesterol levels as well
as increased HDL cholesterol levels and particle size [29]
(Table 1). Interestingly, aortae of mice fed with high-fat diet
protein- (HSP-) 70, endothelial and inducible NOS, HSP-20,
for 6 weeks and human hypercholesterolemic sera showed
NAD-dependent deacetylase sirtuin-1 (Sirt1), and hypoxia-
decreased let-7g expression [24] (Table 1). In the same study
inducible factor 1a [34]. miRNAs are also associated with
a negative feedback regulation has been identified between
the protective effect of ischemic postconditioning against
oxidized LDL receptor 1 and let-7g in primary human aortic
myocardial ischemia/reperfusion injury. Heart specific miR-
smooth muscle cells [24] (Table 1).
1 and miR-133a have been associated with playing a role
Certain microRNAs have been implicated in cellu- in the cardioprotection conferred by ischemic postcondi-
lar responses to oxidative/nitrative stress in cardiovascular tioning through the regulation of apoptosis-related genes
pathologies in preclinical studies [33, 238241]. Many miR- [35, 37]; however, the regulation of miR-1 by ischemic post-
NAs, including miR-21 and miR-199a, have been reported conditioning is controversial [32, 35, 37] (Table 2). Another
to play a role in cardiomyocyte survival during ischemia miRNA, miR-21, has been demonstrated to be implicated in
[33, 242] (Table 1). Moreover, injection of AAV9 vectors ischemic postconditioning, though its role in cardioprotec-
expressing miR-199 and 590 into the peri-infarcted area tion is controversial [35, 36] (Table 2). In addition, loss of
of the myocardium could reduce infarct size and improve the miR-144/451 cluster function has been shown to limit
regeneration after myocardial infarction in mice [243]. We the cardioprotective effect of ischemic preconditioning by
and others have shown that several miRNAs including miR-1, upregulating Rac-1-mediated oxidative stress signaling [38].
miR-21, miR-125b , miR-139-3p, miR-139-5p, miR-181a, miR- Therefore, further preclinical and clinical studies are needed
188, miR-192, miR-212, miR-320, miR-487b, and miR-532 play to investigate the role of miRNAs in ischemia/reperfusion
a role in the mechanism of ischemic preconditioning con- injury and myocardial stress adaptation in healthy and
ferring cardioprotection after ischemia/reperfusion injury diseased conditions, including hypercholesterolemia.
[32, 34] (Table 2). These miRNAs also drive the synthesis miRNAs exert control over diverse metabolic pathways
of important cardioprotective proteins including heat shock and are frequently dysregulated in cardiovascular diseases.
Thus, miRNAs have become a class of promising thera- cardioprotective effects on isoproterenol-induced myocardial
peutic targets. Until miRNA-based therapeutic interventions infarction in rats [253]. Moreover, inhalation of hydrogen
become a reality in clinical medicine many questions should attenuated increased serum cholesterol, cardiac superoxide
be answered in preclinical and clinical studies [244]. Better production, and left ventricular remodeling induced by
technologies and more applicable in vitro and in vivo models intermittent hypoxia in mice [254]. Similarly, consumption of
of human diseases should be developed to identify and hydrogen-rich water beneficially affected serum cholesterol
validate direct mRNA targets of miRNAs [244]. Furthermore, status and oxidative stress markers in patients with potential
improved understanding of the mechanism of action in metabolic syndrome or isolated hypercholesterolemia [255,
each tissue type is necessary. Moreover, development of 256]. These promising results should be confirmed.
organ specific delivery methods for miRNA mimics and
anti-miR oligonucleotides are needed [244]. Nevertheless, 4.3.4. Miscellaneous Examples. Diphenyl diselenide has been
assessment of the efficacy and safety including the analysis recently reported to attenuate hypercholesterolemia-associ-
of the off-target effects of miRNA-based therapeutic tools and ated cardiac oxidative stress and increase antioxidants with-
understanding of the long-term effects of miRNA modulation out affecting plasma level of cholesterol in LDL receptor
in vivo are of key importance in the future [244]. knockout mice [257].
Local infiltration of neuropeptide Y to hypercholes-
4.3.2. Peroxynitrite Scavenging. Peroxynitrite is formed by terolemic swine heart subjected to ischemia was shown to
the rapid reaction of superoxide and NO and is respon- ameliorate cardiac diastolic dysfunction probably by decreas-
sible for a variety of deleterious effects in cardiovascular ing oxidative stress and fibrosis and increasing cell survival in
pathologies [245]. Therefore, development of peroxynitrite the heart [258, 259].
scavengers or compounds catalyzing the decomposition of
peroxynitrite to nontoxic products has been an emerging
field in the last decade [245]. Formation of peroxynitrite 4.4. Physical Activity. A dramatic decrease in an individuals
in the heart of hypercholesterolemic animals has been physical activity is the most obvious change accompanied
demonstrated in various experimental models [6, 8, 246]. by western-type lifestyle and technical development in the
The beneficial effect of decomposition of cardiac peroxyni- industrial countries. Physical inactivity is a risk factor and
trite leading to improved cardiac function in experimental promotes development of civilization diseases. It is widely
hypercholesterolemia was also shown [6, 246]. In isolated accepted that physical activity positively influences a variety
working hearts from Wistar rats or apoB100 transgenic of clinical diseases including obesity, metabolic syndrome,
mice fed with cholesterol-enriched diet, deterioration of dys- and hyperlipidemias, diabetes, and cardiovascular dis-
cardiac function characterized by increased left ventricular eases [260, 261].
end-diastolic pressure (LVEDP) or decreased aortic flow Hypercholesterolemia is accepted as one of the most
was demonstrated, respectively [6, 246]. Pretreatment of important risk factors in the development of different vas-
the animals with the peroxynitrite decomposition catalyst cular and heart diseases. Physical activity and change in
FeTPPS (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato lifestyle are the first choices in normalization of patients
iron (III), chloride) before isolation of the hearts resulted high blood cholesterol level. The mechanisms by which
in an improved LVEDP and aortic flow, respectively [6, physical activity prevents development of metabolic diseases
246]. Whether application of peroxynitrite scavengers would are rather diverse. The primary effect of physical activity
reverse impaired conditioning in hypercholesterolemic ani- can be seen in a metabolic level. Due to higher energy
mals remains to be addressed in future studies. demand, physical activity intensively increases weight loss.
A weight loss of 10 percent can significantly lower the
4.3.3. Hydrogen. Recent advances in basic and clinical risk of cardiovascular diseases by reversing hyperlipidemia.
research have indicated that hydrogen gas is an impor- Moreover, by increasing the catabolic rate of the body,
tant physiological regulatory factor with antioxidant, anti- physical exercise positively influences carbohydrate and lipid
inflammatory, and antiapoptotic protective effects, and thus metabolism and blood lipid profile. Thus, exercise training
the application of molecular hydrogen as a therapeutic med- is associated with increased reliance on lipids as an energy
ical gas in diverse disease conditions has become a feasible substrate, has a systemic lipid-lowering effect, and results
therapeutic strategy [247]. Hydrogen is suggested to be an in remodeling of skeletal muscle lipid metabolism toward
efficient, nontoxic, highly bioavailable, and low-cost antiox- increased oxidation and neutral lipid storage and turnover
idant supplement for patients with pathological conditions [262]. Physical activity has substantial effects on the liver
involving ROS-induced oxidative stress [248]. Therapeutic metabolism as well, modifying lipoprotein levels to a more
hydrogen can be applied by different delivery methods healthy composition. These effects can be enhanced by
including inhalation of hydrogen gas, drinking hydrogen using concurrent dietary restrictions; for example, a low-
dissolved in water, and injection with hydrogen-saturated cholesterol diet definitely helps to regulate lipid profile in
saline [247]. In the heart, hydrogen attenuated doxorubicin- the body. Regular participation in physical activity as well
induced heart failure in rats [249], cardiac dysfunction in as a single exercise session can positively alter cholesterol
streptozotocin-induced diabetic mice [250], rat cardiac cold metabolism [263]. Exercise is involved in increasing the
ischemia/reperfusion injury [251], and left ventricular hyper- production and action of several enzymes that function to
trophy in spontaneous hypertensive rats [252] and exerted enhance the reverse cholesterol transport system [263].
Nevertheless, physical activity has secondary effects on difficult. One possible answer is that during exercise an
the prevention of development of metabolic diseases by, intensive but only a temporary increase of oxidative stress
for example, modifying oxidative stress. Physical activity is occurs resulting in a possibility for cardiac adaptation by an
believed to be a protective modulator of oxidative stress improved enzymatic and nonenzymatic antioxidant capacity,
in hyperlipidemia; however, high intensity physical exercise cytoprotection, aerobic capacity, training-induced muscular
itself can definitely increase oxidative stress in patients [264 adaptation, mitochondrial biogenesis, and so forth [268]. In
266]. Two key free radicals are the most important during hypercholesterolemia with no exercise, the continuously ele-
physical activity, that is, superoxide and NO. The exact vated oxidative/nitrative stress, however, does not allow the
sources of these radicals are not fully known; however, completion of cardioprotective mechanisms. End-effectors
mitochondria are often cited as the predominant source of of cardioprotection involve the activation of ATP-sensitive
ROS in muscle cells. Investigators have often assumed that the K+ -channels (KATP). We have recently demonstrated that a
increased ROS generation that occurs in muscle fibers during cholesterol-enriched diet inhibited cardioprotection induced
contractile activity is directly related to the elevated oxygen by KATP activators and that cholesterol diet may impair
consumption. However, growing evidence argues against cardiac KATP channels [282]. It is well accepted that the
mitochondria being the dominant source of ROS production opening of KATP channels generates ROS; however, an
in skeletal muscle during exercise [264]. Another possible ambient oxidative state also modifies redox-sensitive KATP
source is NADPH oxidase, which is normally quiescent, channels, as superoxide, hydrogen peroxide, and peroxyni-
but when it becomes activated, during muscle contraction trite open KATP channels in the heart. These results show
or when recruited for antimicrobial and proinflammatory that increased oxidative stress interferes with KATP channel
events, it can generate large amounts of superoxide [264, 267, function and therefore might explain why cardioprotection is
268]. Since the discovery that contracting skeletal muscles lost in hyperlipidemia.
produce ROS, many investigators have assumed that skeletal Increased physical activity can be also mimicked in exper-
muscle provides the major source of free radical and ROS imental animal models by applying ventricular overdrive
generation during exercise. Nonetheless, other tissues such pacing of the heart, a method that was reported to induce
as the heart, lungs, or blood may also contribute to the total both preconditioning and postconditioning by increasing the
body generation of ROS during exercise [264]. Thus the oxygen demand (relative hypoxia) instead of limiting oxygen
increased catabolic process together with multiplied oxygen supply (absolute hypoxia) in isolated heart models [283
consumption in both skeletal and cardiac muscles during 285]. Peroxynitrite plays an important role in different con-
exercise exacerbates superoxide production. ditionings induced by either ischemia or ventricular pacing
The other major free radical which contributes to oxida- [11, 286]. It is known that experimental hypercholesterolemia
tive/nitrative stress is NO. NO is responsible for the relaxation blocks the cardioprotective effect of postconditioning at least
of vessels [269] and plays an important role in matching tissue in part via deterioration of postconditioning-induced early
perfusion to demand [270]. The release of nitric oxide by increase in peroxynitrite formation during reperfusion [11].
the endothelial cell can be upregulated by exercise [261, 271]. Thus one can speculate that regular physical exercise is able
However, hypercholesterolemia impairs endothelial function to restore the protective effects of pre- or postconditioning in
(e.g., the NO-cyclic GMP-phosphodiesterase 5 pathway), hypercholesterolemia via modifying cardiac oxidative stress
limits shear stress-induced vasodilation, and is therefore and by beneficially altering lipid profile in the blood.
expected to reduce exercise-induced vasodilation [272]. In addition to emphasizing the importance of regular
In addition to the modulation of ROS and RNS pro- exercise, novel future directions have been implicated to take
duction, physical exercise may also affect the antioxi- advantage of exercise-induced benefits. Thus development of
dant defense systems. McCommis et al. have demonstrated new exercise mimetics has a promising role in the future for
that familial hypercholesterolemia reduces mitochondrial treatment of patients [287].
antioxidants, increases mitochondrial oxidative stress, and
enhances the mitochondrial permeability transition response
5. Conclusions
in the porcine myocardium [19]. They also showed that
exercise training can reverse these detrimental alterations Oxidative and nitrative stress has been implicated as a
without altering serum cholesterol level [19]. pathophysiological mechanism of cardiovascular diseases;
In spite of the extensive research, the cardioprotective however, there is still no breakthrough regarding the use
effect of exercise which is mediated by redox changes of general antioxidant therapies in clinical practice. The
is still a question of debate. Several studies showed that possible reasons for these disappointing results and some
hyperlipidemia impairs exercise capacity itself [273] or promising aspects of potential antioxidant therapy have been
the effect of physical activity [272, 274, 275]. However, discussed in detail recently [288]. Although hypercholestero-
a number of exercise programs have effectively reversed lemia occurs frequently in the adult population, the number
hypercholesterolemia-induced changes mainly within the of publications investigating myocardial oxidative/nitrative
vasculature by improving NO bioavailability in both animal stress and its cardiac consequences is relatively limited
studies and humans [276281]. especially in humans. This is unfortunate, as patients with
The exact explanation why physical activity, which simi- hypercholesterolemia are at an increased risk for severe
larly to hyperlipidemia leads to an increased oxidative stress, pathological conditions such as myocardial infarction and
is able to protect the heart in hypercholesterolemia is rather heart failure and hypercholesterolemia has been shown
to interfere with endogenous cardioprotective mechanisms. [8] Z. V. Varga, K. Kupai, G. Szucs et al., MicroRNA-25-
Therefore, finding proper approaches to beneficially affect dependent up-regulation of NADPH oxidase 4 (NOX4) medi-
hypercholesterolemia and its myocardial consequences is ates hypercholesterolemia-induced oxidative/nitrative stress
crucial. These reasons warrant further preclinical and clinical and subsequent dysfunction in the heart, Journal of Molecular
studies to better understand the pathological events in the and Cellular Cardiology, vol. 62, pp. 111121, 2013.
heart relating to hypercholesterolemic conditions and to [9] R. M. Osipov, C. Bianchi, J. Feng et al., Effect of hypercholes-
find the best approaches to interact. Moreover, many of terolemia on myocardial necrosis and apoptosis in the setting of
the potential modulators of oxidative/nitrative stress require ischemia-reperfusion, Circulation, vol. 120, no. 1, pp. S22S30,
further development to optimize their effects for application 2009.
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The authors declare that there is no conflict of interests [11] K. Kupai, C. Csonka, V. Fekete et al., Cholesterol diet-
regarding the publication of this paper. induced hyperlipidemia impairs the cardioprotective effect of
postconditioning: role of peroxynitrite, The American Journal
of PhysiologyHeart and Circulatory Physiology, vol. 297, no. 5,
Acknowledgments pp. H1729H1735, 2009.
[12] B. Halliwell, Biochemistry of oxidative stress, Biochemical
This work was supported by the European Union, cofi- Society Transactions, vol. 35, part 5, pp. 11471150, 2007.
nanced by the European Social Fund, within the framework
[13] W. Droge, Free radicals in the physiological control of cell
of TAMOP-4.1.1.C-13/1/KONV-2014-0001 project, and by
function, Physiological Reviews, vol. 82, no. 1, pp. 4795, 2002.
grants from the Hungarian Scientific Research Fund (OTKA
K 115990). Csaba Csonka was supported by the Janos Bolyai [14] G. F. Kocsis, T. Csont, Z. Varga-Orvos, L. G. Puskas, Z.
Research Scholarship of the Hungarian Academy of Sciences. Murlasits, and P. Ferdinandy, Expression of genes related to
The authors thank Jeremy Parrott Ph.D. language editor for oxidative/nitrosative stress in mouse hearts: effect of precondi-
tioning and cholesterol diet, Medical Science Monitor, vol. 16,
proofreading the paper.
no. 1, pp. BR32BR39, 2010.
[15] L. G. Puskas, Z. B. Nagy, Z. Giricz et al., Cholesterol diet-
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http://dx.doi.org/10.1155/2016/7409196
Research Article
Apocynin and Diphenyleneiodonium Induce
Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk
Activity in Mouse Embryonic Stem Cells
Jan KuIera,1 Lucia Bin,1,2,3 Katelina tefkov,1 Josef Jaro,3 Ondlej VaIek,2,4
Josef VeIela,1 Luk Kubala,2,4 and Jil Pachernk1,4
1
Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 267/2, 61137 Brno, Czech Republic
2
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 2590/135, 61200 Brno, Czech Republic
3
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 62500 Brno, Czech Republic
4
International Clinical Research Center, Center of Biomolecular and Cellular Engineering, St. Annes University Hospital,
Pekarska 53, 65691 Brno, Czech Republic
Correspondence should be addressed to Jir Pachernk; jipa@sci.muni.cz
Academic Editor: Tomris Ozben
Copyright 2016 Jan Kucera et al. This is an open access article distributed under the Creative Commons Attribution License,
Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to
influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin
and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to
NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore
we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells.
Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin
inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of
canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without
affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling
pathways.
1. Introduction to regulation of different processes including proliferation,

migration, differentiation, immunomodulation, and oxygen
Reactive oxygen species (ROS) play multiple roles in the biol- sensing, and therefore, its expression and activity are tissue
ogy of the cell [1]. NADPH oxidase (NOX) and oxidative reac- specific and are tightly controlled [4, 5]. On the basis of the
tions on the mitochondrial membrane are the main sources homology to the catalytic subunit of the original phagocytic
of ROS, although it can also be produced by other enzymatic NOX (gp91phox or preferably NOX2), other NOX isoforms
and nonenzymatic sources [2, 3]. NOX is a membrane- NOX1, NOX3, NOX4 and NOX5have been identified in
bound protein complex generating superoxide anion (O2 ) nonphagocytic cells. In parallel, two other members of the
from molecular oxygen which initiates the cascade of free NOX family were discovered, namely, dual oxidases 1 and 2
radical reactions in response to various stimuli. Production (DUOX 1, 2), initially also referred to as thyroid oxidases [6].
of ROS by NOX family has long been considered a unique Despite studies suggesting the importance of NOXs in
property of phagocytic cells, which utilize this enzyme as a general, the involvement of individual NOX family members
part of host defense immune system. Currently, the regulated in specific function is still not completely understood. One of
ROS production in nonphagocytic cells by NOX was linked the reasons is a lack of highly specific inhibitors that would
reliably block particular NOX. The most frequently used DPI enhances both PI3K and Erk activity. Interestingly, APO
inhibitors employed in experiments are vanillin derivative 4- strengthens Wnt activity, pointing out another unknown
hydroxy-3-methoxyacetophenone (trivial names: apocynin mechanism of APO-mediated changes in signaling cascades
or acetovanillone, APO) and diphenyleneiodonium chloride regulating mES. In contrast to PI3K and Erk, we did not
(DPI). Both of these drugs were applied in numerous in vitro observe any effect of APO or DPI on Stat3 phosphorylation,
and in vivo studies and although their effect was attributed which is considered to play the major role in mES mainte-
primarily to NOX inhibition, specificity of APO and DPI nance.
remains questioned [7].
The proposed molecular mechanism of APO-mediated
NOX inhibition is not fully understood but it involves impair- 2. Material and Methods
ment of NOX complex assembly and activation [8]. APO was
also shown to act directly as ROS scavenger [9]. Contrarily to 2.1. Cell Culture and Treatment. A feeder-free adapted mES
this finding, other studies suggest that APO is rather a proox- line R1 was propagated as described previously [29] in an
idant stimulating ROS production [1012]. Further, APO was undifferentiated state by cell culturing on tissue culture
also shown to modulate the generation of arachidonic acid- plastic coated by gelatin (0.1% porcine gelatin solution in
derived inflammatory mediators [13]. water) in Dulbeccos modified Eagles medium (DMEM)
DPI was reported not only to affect NOX, but also to containing 15% fetal bovine serum (FBS), 100 IU/mL peni-
interfere with other flavoenzymes, including nitric oxide cillin, 0.1 mg/mL streptomycin, 1x nonessential amino acid
synthase and xanthine oxidase [14, 15]. Inhibitory effects of (all from Gibco-Invitrogen, UK), and 0.05 mM -mercap-
DPI on mitochondrial ROS production were also shown [16]. toethanol (Sigma-Aldrich, USA), supplemented with 5 ng/
On the other hand, DPI induces O2 mediated apoptosis mL of leukemia inhibitory factor (LIF, Chemicon, USA) re-
[17], inhibits cell redox metabolism, and promotes general ferred to here as the complete medium.
oxidative stress [18]. DPI is also suggested as a nonselective APO, DPI, hydrogen peroxide (H2 O2 ), N-acetylcysteine
blocker of ionic channels [19]. The above mentioned nonspe- (NAC), and LY294002 (LY) were provided from Sigma-
cific effects are thought to be responsible for contradictory Aldrich. Stock solutions of APO (0.4 M), DPI (10 mM), and
results obtained using these inhibitors. Further studies are LY (10 mM) were prepared by dissolving the compounds in
needed to better understand the actions of APO and DPI not dimethyl sulfoxide. Aliquots were stored at 20 C. NAC was
directly related to NOX inhibition. prepared as a 0.5 M stock solution in serum-free DMEM
Intracellular formation of ROS leading to overall redox medium, pH was adjusted to 7.4, and filter-sterilized aliquots
status modulation is important for regulation of pluripotent were stored at 20 C. Drugs were added directly to the incu-
cells differentiation [20]. In mouse embryonic stem cells bation medium or freshly prediluted in sterile phosphate-
(mES), pluripotent cells derived from inner cell mass of buffered saline (PBS) to desired concentration.
blastocyst, redox alterations are thought to play a role in the
balance between self-renewal and differentiation. Undifferen-
tiated mES have several times lower ROS level in comparison 2.2. Cell Proliferation. The cell proliferation was determined
with differentiating mES [21]. It was shown that short term by estimation of overall cellular protein mass in whole
increase of the ROS favors differentiation into cardiomy- cell lysates that reflects the cell number as demonstrated
ocytes [22, 23] and into endoderm and mesoderm lineage previously [34]. ES cells were seeded to 24-well plate in
[24]. complete media at density 5 000 cells per cm2 . Next day, the
Several signaling pathways are crucial for the regulation cells were treated with drugs for further 48 hours. Finally, the
of self-renewal and differentiation of mES. Primarily, the mES cells were washed twice with PBS and lysed in SDS buffer
pluripotency is controlled by Stat3 together with PI3K/Akt (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 1%
signaling pathways [2527]. Importance of PI3K signaling SDS, 1 mM EDTA). Protein concentration was determined
was demonstrated in many studies where its inhibition using DC protein assay (Bio-Rad, USA) kit according to
negatively regulates the self-renewal in mES [28, 29]. Further, manufacturers instructions.
MAPK/Erk signaling pathway is rather important for mES
differentiation as its inhibition improves maintenance of
the pluripotent stem cell phenotype [30]. A growing body 2.3. Determination of NOX Expression. Total RNA was
of evidence indicates that Wnt/-catenin signaling pathway extracted using the UltraClean Tissue & Cells RNA Isolation
plays a vital role in the regulation of mES fate [31]. Signaling Kit (MO BIO Laboratories, USA) for ESC and RNAzol RT
pathways including Stat3, PI3K/Akt, Erk, and Wnt are mod- (Molecular research center Inc., USA) for mouse tissues
ified by ROS production [5, 32, 33] and therefore might be according to manufacturers instructions. 1 g of total RNA
affected by NOX or other ROS modulating agents, although was used for cDNA synthesis with DyNAmo cDNA Syn-
the importance of this phenomenon in mES remains elusive. thesis Kit (Finnzymes, Finland) according to the manufac-
Our study demonstrates unexpected prooxidant activity turers instructions. qPCR was performed in LightCycler 480
of APO and DPI in undifferentiated mES together with instrument using LightCycler Probes Master with Universal
impairment of cell proliferation. Further, we show that APO Probe library probes (all from Roche, Germany) accord-
inhibits PI3K signaling accompanied by decrease in protein ing to manufacturers instructions. Ribosomal protein L13A
level of critical ES pluripotency regulator Nanog, whereas (RPL13A) was used as reference gene; data are presented as
2(Cq(target)Cq(reference)) . The primers and probes used were as 2.6. Western Blot Analysis. Western blot analysis, cell sample
follows: harvesting, and preparation were performed by a standard
NOX1: 5 tggattttctaaactaccgtctcttc, 5 caaagtttaatgctgcat- procedure as presented previously [29]. We used the fol-
gacc3 , #20; NOX2: 5 tgccaacttcctcagctaca3 , 5 gtgcacagc- lowing primary antibodies against Nanog, -actin (Abcam,
aaagtgattgg3 , #20; NOX3: 5 tgaacaagggaaggctcatt3 , 5 cat- USA), p-Akt (S473), Akt, p-Stat3 (Y705), Stat3, Erk1/2, p-
cccagtgtaaagctatgtga3 , #20; NOX4: 5 catttgaggagtcactgaact- Erk1/2 (T202/Y204), and p-GSK3 (S9) (all from Cell Sig-
atga3 , 5 tgtatggtttccagtcatccag3 , #5; DUOX1: 5 acagatggg- naling Technology, USA). Following immunodetection, each
gcagcaaag3 , 5 gctggtagacaccatctgcat3 , #20; DUOX2: 5 cag- membrane was stained by amido black to confirm the transfer
acagctttttgctcaggt3 , 5 cacttgctgggatgagtcc3 , #64; RPL13A: of the protein samples. The total level of -actin was detected
5 catgaggtcgggtggaagta3 , 5 gcctgtttccgtaacctcaa3 , #25. as loading control.
2.4. Analysis of ROS Production in Live Cells by Automated 2.7. Cell Transfection and TOPflash Luciferase Reporter Assay.
Time-Lapse Image (Live Imaging Fluorescent Microscopy). Cells were transiently transfected using polyethyleneimine in
The cells were seeded to 96-well plate designed for live imag- a stoichiometry of 4 L per 1 g of DNA. Super8X TOPflash
ing fluorescence microscopy (Greiner Bio-one, Germany). construct, Renilla luciferase construct, and expression vec-
After 24 hours, cells were pretreated with tested drugs for 15 tor for mutant nondegradable -catenin, and S33--catenin
minutes and loaded with hydroethidine (HE, 5 M, Sigma- (codon 33 substitution of Y for S, generously provided by
Aldrich). Live imaging of prepared samples was performed at Professor Korswagen) were used in concentration of 0.5 g
37 C and 5% CO2 atmosphere using the high-content screen- per well in a 24-well plate 24 hours after seeding. 6 hours
ing microscope ImageXpressMicroXL (Molecular Devices, after transfection medium was changed and cells were treated
USA). Seven images per well were acquired during 120 with NOX inhibitors or LY for 24 hours. For cell stimulation
minutes with scanning interval of 10 minutes. By Gaussian Wnt3a or control conditioned medium [41] was added 8
thresholding of the HE fluorescence images, mask area of hours before harvest. Dual-Luciferase assay kit (Promega,
the viewfield covered with metabolizing cells was detected USA) was used according to the manufacturers instructions
using the Otsu method of fitting individual objects. The for the evaluation of luciferase activity. Relative luciferase
10640 images for every measured plate were analyzed with units were measured on a plate luminometer Chameleon V
CellProfiler [35], running on processor cluster provided by (Hidex, Finland) and normalized to the Renilla luciferase
MetaCentrum (The National Grid Infrastructure). Intensity expression.
of fitted regions was lowered by minimal intensity of an
appropriate image to subtract the background and the mean 2.8. Statistical Analysis. Data are expressed as mean +
intensity per individual well was then calculated and visu- standard error in the mean (SEM). Statistical analysis was
alized with Knime [36], using HCS tools and R Statistics assessed by -test or by one-way analysis of variance ANOVA
Integration extensions [37]. and Bonferronis Multiple Comparison posttest. The values of
< 0.05 were considered statistically significant ( < 0.05,

2.5. High-Performance Liquid Chromatography (HPLC) Anal- < 0.01, and < 0.001).
ysis of ROS Production. The HPLC detection of O2
was based on the detection of a specific product 2- 3. Results
hydroxyethidium 2-OH-E(+) which is formed in the reaction
of O2 with HE [38, 39]. Besides specific 2-OH-E(+), also a 3.1. Expression of NOXs in mES Is Relatively Low. To confirm
nonspecific product of hydride acceptors with HE ethidium the assumption of the negligible presence of NOX and DUOX
(E+) was detected. The cells were seeded to 6-well plate and homologues in the undifferentiated mES, the gene expression
before the treatment the medium was changed for DMEM was compared to the selected mouse tissues. Particular
(without phenol red and sodium pyruvate) with 1% FBS. The organs were chosen based on the described presence of NOX
cells were treated with APO and DPI for 120 minutes in total; homologues by various authors [4, 6]. In agreement with the
30 minutes before the end of the experiment HE in final literature, all determined NOX homologues NOX1, NOX2,
concentration 10 M was added. The medium after centrifu- NOX3, NOX4, DUOX1, and DUOX2 showed two to three
gation was stored for optional HPLC analysis. To extract the orders lower expression in undifferentiated mES compared
HE products, ice cold methanol was added to the cells [40] to selected tissues (Figures 1(a)1(f)). The comparison of the
for 15 minutes at 4 C in dark, shaking. The supernatant was NOXs relative expression in mES revealed the NOX4 expres-
transferred to an Eppendorf tube and centrifuged. A 75 L sion to be the highest, approximately 10 times compared to
sample was injected into the HPLC system (Agilent series other NOXs (Figure 1(g)), altogether, it can be concluded that
1100) equipped with fluorescence and UV detectors (Agilent the expression of all NOXs except NOX4 is very low.
series 1260) to separate the 2-OH-E(+) product. Fluorescence
was detected at 510 nm (excitation) and 595 nm (emission). 3.2. APO and DPI Affect mES Proliferation. Both APO and
The mobile phase consisted of H2 O/CH3 CN. Kromasil C18 DPI affected growth of mES in dose-dependent manner in
(4.6 mm 250 mm) column was used as the stationary phase. the concentration range 0.1, 0.25, 0.5, 1.0, and 2.0 mM for
Elution conditions for the analysis of HE and its products APO (Figure 2(a)) and 10, 20, 40, 80, 160, and 320 nM for
were used from Nature Protocols [38]. DPI (Figure 2(b)). Data showed decrease in cell proliferation
NOX1 NOX2
0.0008 0.05
0.04
0.0006
Relative mRNA expression

0.03
0.0004
20x 0.02
900x
0.0002
0.01
0.0000 0.00
Heart mES Spleen mES
(a) (b)
NOX3 NOX4
0.010 0.5
0.008 0.4
0.006 0.3
0.004 0.2
300x 850x
0.002 0.1
0.000 0.0
Kidney mES Kidney mES
(c) (d)
DUOX1 DUOX2
0.0015 0.0008
0.0006
0.0010
0.0004
0.0005
7x
0.0002 60x
0.0000 0.0000
Lung mES Lung mES
(e) (f)
Figure 1: Continued.
NOXs in mES
0.0006

0.0004
0.0002
0.0000
NOX1 NOX2 NOX3 NOX4 DUOX1 DUOX2
(g)
Figure 1: Relative expression of NOX1 (a), NOX2 (b), NOX3 (c), NOX4 (d), DUOX1 (e), and DUOX2 (f) mRNA in mES and selected tissues.
Comparison of relative expression of individual NOXs and DUOXs mRNA within mES cells is also shown (g). Data are presented as mean +
SEM from at least two independent experiments.
APO DPI
150 150
Cell growth (% of ctr)
Cell growth (% of ctr)
A A
A A
100 100 A
B
C B
50 50
BC BC BC
D C
0 0
Ctr 0.1 0.25 0.5 1 2 Ctr 10.0 20.0 40.0 80.0 160.0 320.0
(mM) (nM)
(a) (b)
Figure 2: Effect of APO (a) and DPI (b) on mES proliferation after 48 h treatment based on total cellular protein mass. Data represent mean
+ SEM from four independent experiments. Statistical significance was determined by ANOVA post hoc Bonferronis Multiple Comparison
test, < 0.05. The groups marked differently by symbol letters are statistically significantly different from each other.
using concentration from 0.5 mM (APO) and from 20 nM (Figures 3(d) and 3(e)). This analysis shows potentiation of
(DPI) after 48-hour treatment. IC50 of APO and DPI for mES nonspecific HE oxidation rather than O2 formation in DPI
determined from these data were 1.1 0.2 mM and 18.5 treated cells, but not in APO treated cells.
3.2 nM, respectively.
3.4. Effect of APO and DPI on Stat3, Akt, and Erk Phosphoryla-
3.3. APO and DPI Do Not Inhibit but Potentiate Formation tion in mES. To investigate the effect of short term treatment,
of ROS in mES. Despite the very low expression of NOXs mES were serum and LIF starved for 12 hours and treated
enzymes in undifferentiated mES, the ROS production was by APO and DPI for 20 minutes followed by 20-minute
detectable in our mES lines. Contrary to the expectations, stimulation with FBS or LIF. APO treatment resulted in
treatment by APO or DPI significantly induced generation decrease of Akt phosphorylation in every condition without
of ROS in mES cells detected by live imaging analysis effect on Erk. DPI had no significant effect on Akt and Erk
of HE fluorescence, continuously for up to 120 minutes kinases signaling. Level of p-Stat3 remained unchanged by
(Figures 3(a), 3(b), and 3(c)). To confirm the specificity NOX inhibitors (Figure 4).
of this determination, the formation of specific product of To further clarify the dose-dependent effect of tested
HE reaction with O2 , the 2-OH-E(+), and also nonspe- drugs, we employed the same experimental design with
cific product, ethidium (E+), were determined by HPLC serum starvation and FBS activation. Moreover, glutathione
5 5
4 4
Relative fluorescence
3 3
2 2
1 1
0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
(minutes) (minutes)
Ctr Ctr
APO DPI
(a) (b)
3 1.5 2-OH-E(+)

Relative units (area of peak)

2 1.0
1 0.5
0 0.0
Ctr APO DPI Ctr APO DPI
(c) (d)
2.5 E(+)

2.0
Relative units (area of peak)
1.5
1.0
0.5
0.0
Ctr APO DPI
(e)
Figure 3: ROS production in mES cells treated by 1 mM APO and 100 nM DPI computed from the automated time-lapse image acquisition of
HE fluorescence for 120 minutes (a, b); selected single time point 60 minutes (c). HPLC determination of specific 2-OH-E(+) and nonspecific
product E(+) of HE oxidation in the presence of 1 mM APO and 100 nM DPI (d, e). Data are presented as mean + SEM from four independent
experiments. Statistical significance was determined by ANOVA post hoc Bonferronis Multiple Comparison test, < 0.05. The groups
marked by an asterisk are statistically significantly different from control.
SF LIF FBS Nanog protein level in mES, independently of NAC sup-

Ctr APO DPI Ctr APO DPI Ctr APO DPI plementation (Figure 6). Level of Stat3 phosphorylation was
modulated in the above mentioned experimental condition
p-Stat3
by neither APO nor DPI treatment (data not shown). We did
not observe any effect of H2 O2 on the evaluated signaling
Stat3
pathways after 24 hours (data not shown).
3.5. APO Augments Canonical Wnt Activity in mES. To

p-Akt assess the effects of APO and DPI supplementation on activ-
ity of Wnt pathway, we used TCF/LEF reporter gene assay
(TOPflash) to determine the level of canonical Wnt activation
Akt
mediated by -catenin which specifically induces the tran-
scriptional activity of TCF/LEF [42]. Firstly, we examined
p-Erk our system with addition of Wnt3a conditioned media or
exogenous nondegradable -catenin, both known agonists,
to promote its activation. These interventions induced tran-
Erk scription activity of reporter gene 45 and 25 times, respec-
tively (Figure 7(a)).
PI3K inhibitor LY294002 (LY) and both NOXs inhibitors
-actin APO and DPI did not change the spontaneous -catenin
mediated transcription activity of TCF/LEF (Figure 7(b)). In
Figure 4: Effect of APO (2 mM) and DPI (100 nM) on the Stat3, Akt, contrast, when the cells were treated by Wnt3a conditioned
and Erk phosphorylation in serum starved mES cells (SF) treated media, presence of LY and APO significantly augmented the
by drugs for 20 minutes followed by LIF (5 ng/mL) or FBS (15%) TCF/LEF transcription activity (Figure 7(c)). On the other
stimulation for 20 minutes. Total level of -actin was used as a hand, DPI had no effect (Figure 7(c)). However, all tested
loading control. A typical representative western blot is shown. drugs did not have any effect in the presence of exogenous
nondegradable -catenin (Figure 7(d)).
To further clarify the effects of LY, APO, and DPI on Wnt/
precursor NAC and H2 O2 were used as a bona fide antiox- -catenin signaling, the GSK3 S9 phosphorylation, allowing
idant and a prooxidant, respectively, to distinguish between accumulation of -catenin, was evaluated [43]. Treatment
effects mediated via redox changes in cultivated mES and with both APO and LY but not DPI decreased GSK3 S9
ROS independent actions of APO and DPI. phosphorylation in this setup as shown by the western blot
APO abolished phosphorylation of Akt in a dose- analysis (Figure 7(e)).
dependent manner with supportive effect on phosphory-
lation of Erk in higher concentrations. In contrast, DPI 4. Discussion
slightly increased phosphorylation of Akt and also upregu-
lated phosphorylation of Erk in the highest concentration. APO and DPI are the most commonly used inhibitors of
Consistent with expectations, H2 O2 increased both Akt NOX, involved in numerous studies despite the increasing
and Erk phosphorylation. NAC had no effect on signaling evidence questioning their specificity. We employed mES as a
in this setup (Figure 5(a)). To test whether the effect on model to analyze effects of these compounds that might not
signaling was also preserved during cultivation in complete be directly mediated through impairment of NOXs, because
medium, mES were treated by the same concentration of of their generally negligible expression in undifferentiated
drugs for 1 hour. In this case, similarly to previous treatment, mES. We aimed to investigate modulation of ROS production
APO inhibited Akt phosphorylation in a dose-dependent by APO and DPI in mES as well as interactions with signaling
manner. Erk phosphorylation was impaired in the presence pathways important for stem cell regulation.
of the highest concentration of APO. Notably, in this setup Although NOXs were attracting attention as an important
100 M concentration of DPI strongly induced both Akt and source of intracellular ROS production for a long time, their
Erk phosphorylation. Contrary to the previous setup, NAC role in stem biology remains poorly understood. Previously,
decreased phosphorylation of both pathways (Figure 5(b)). it was demonstrated that NOXs expression is precisely
Finally, we examined the impact of APO and DPI on regulated during embryonic stem cell differentiation into
Akt and Erk activity after 24 hours in absence or presence of cardiomyocytes [44, 45] and vascular smooth muscle lineage
NAC (Figure 6). Contrary to the effect observed after a short [46].
term treatment, Akt phosphorylation was upregulated when In our experiments, we observed generally low level of
APO was employed. Phosphorylated form of Akt was also NOXs/DUOXs expression close to the limit of detection,
increased following the DPI treatment. This activation could which we concluded both from real Cq values of PCR amplifi-
be prevented by addition of NAC. Erk phosphorylation was cation and from comparison of NOXs/DUOXs expression in
slightly decreased by APO treatment and augmented by DPI several employed tissues. As such control sample, tissues with
even in the presence of NAC. APO strongly downregulated well described NOXs/DUOXs expression and activity were
Ctr APO DPI NAC H2 O2 Ctr APO DPI NAC H2 O2
p-Akt p-Akt
Akt Akt
p-Erk p-Erk
Erk Erk
-actin -actin
(a) (b)
Figure 5: Effect of APO (1, 2, 4 mM), DPI (0.1, 1, 100 M), NAC (5, 10, 20 mM), and H2 O2 (0.5, 1 mM) on the Akt and Erk phosphorylation
in serum starved mES cells treated by drugs for 20 minutes followed by 15% FBS stimulation for 20 minutes (a) and cells treated for 1 hour in
complete medium (b). Total level of -actin was used as a loading control. A typical representative western blot is shown.
NAC control tissue (kidney). NOX4 was shown to be constitutively

Ctr APO DPI Ctr APO DPI active, and hence its activity is mostly regulated by the level
of its expression [49]. Concerning other potential sources of
p-Akt intracellular ROS, it is also noteworthy that favored reliance
on anaerobic glycolysis in undifferentiated mES leads to
reduced mitochondrial biogenesis and activity, manifested
by declined ROS production [50, 51]. Thus mES can be
Akt considered not only NOX-low, but also overall ROS-low
model.
Next, we aimed to assess effect of APO and DPI on pro-
liferation of undifferentiated mES. Selection of used concen-
p-Erk trations was based on comprehensive literature search. APO
as NOX inhibitor was used in range 301200 M [9, 52] with
the upper range of these concentrations corresponding to
APO supplementation preferably employed in our experi-
Erk ments. Regarding DPI, many authors used concentrations
ranging 1100 M {summarized in [53]} which is approxi-
mately one order of magnitude higher than doses used in our
experiments, as we observed significant growth impairment
Nanog even when concentrations as low as 20 nM were applied.
Impact on cell proliferation after NOX inhibitors treatment
was described earlier, for both normal and transformed cell
-actin
lines. The observed effects of DPI and APO were suggested to
be attributed to the various mechanisms including changes in
NOX mediated ROS production, downregulation of integrin
Figure 6: Effect of APO (1 mM) and DPI (10 nM) in absence expression, cell cycle arrest, or modulation of mitogenic-
and presence of antioxidant NAC (10 mM) on the Akt and Erk, signaling pathways [5457]. Therefore, we can assume that
phosphorylation and Nanog protein level in mES cells after 24 hours direct modulation of ROS production could contribute to the
in complete medium. Total level of -actin was used as a loading observed decrease of mES proliferation. At the same time, the
control. A typical representative western blot is shown. effects of inhibitors employed in this study can also be related
to their direct effects on the other promitogen cell signaling
pathways as discussed later. The potential of APO and DPI to
inhibit cell proliferation could also be beneficial in the context
used [4, 6]. Among NOXs and DUOXs, NOX4 expression of anticancer agents. A recent publication showed that APO
was the highest in mES, which is in agreement with other suppressed prostate cancer and that the reduction of Rac1 and
authors [46, 47]. It may also correspond to relative abundant NFB phosphorylation was involved [58].
expression of this enzyme across other tissues [48]. However, Further, we assessed how NOX inhibitors affected ROS
it should be emphasized that level of NOX4 transcript in production in our model. Due to their natural short half-
mES was still nearly three orders below expression in selected life and high reactivity, precise detection of ROS represents
60 2.0

Relative luminescence
1.5
40

1.0
20
0.5
0 0.0
Ctr Wnt3a S33--catenin Ctr LY APO DPI
(a) (b)
2.0 2.0

1.5 1.5
1.0 1.0
0.5 0.5
0.0 0.0
Ctr LY APO DPI Ctr LY APO DPI
(c) (d)
Wnt3a S33--catenin
APO
APO
APO
DPI
DPI
DPI
Ctr
Ctr
Ctr
LY
LY
LY
p-GSK3
-actin
(e)
Figure 7: Effect of APO and DPI on Wnt pathway activity determined by TOPflash assay and GSK3 phosphorylation on S9. Effect of the Wnt3a
conditioned media or exogenous nondegradable -catenin on the transcriptional activation of a reporter gene (a). Effect of LY (10 M), APO
(1 mM), and DPI (10 nM) on the spontaneous (b), Wnt3a conditioned media induced (c), and exogenous nondegradable -catenin-induced
transcriptional activation (d). Data represent mean + SEM, from at least four independent experiments. Statistical significance was determined
by ANOVA post hoc Bonferronis Multiple Comparison test ( < 0.05, < 0.01, and < 0.001). Effect of LY, APO, and DPI on GSK3
(S9) phosphorylation (e). Total level of -actin was used as a loading control. A typical representative western blot is shown.
a tremendous challenge, especially in nonphagocytic cells. dimeric products [38, 59, 60]. Despite the expectations, the
We were aware of limits in ROS measurement with respect live imaging assay showed significant prooxidant activity
to specificity and generation of possible artefacts; thereof APO and DPI when continuous nonoscillating probe
fore, we used two different assays, live imaging fluorescent oxidation was determined by live imaging. On the other
microscopy and HPLC, both utilizing ROS-sensitive probe hand, HPLC analysis used for determination of specific HE
HE. The reaction between O2 and HE generates a highly derivatives did not confirm O2 production after APO and
specific red fluorescent product 2-hydroxyethidium 2-OH- DPI treatment. The only observed effect was significant DPI-
E(+). However, in the intracellular milieu, the presence of mediated elevation of E(+). APO did not induce generation
redox metal ions or hemeproteins with peroxidase activity of HE oxidation products {both 2-OH-E(+) and E(+)}, which
or other one-electron oxidants can oxidize HE to several is partially in contrast with the results from live imaging
nonspecific products, including the ethidium E(+) and fluorescent microscopy where all nonspecific HE oxidation
fluorescent products are summarized [60]. This suggests that pathways. In agreement with other studies, both Akt and Erk
other oxidants, rather than O2 , are responsible for increase kinases responded in ROS-sensitive manner [67, 68], which
in HE fluorescence. we demonstrated by H2 O2 -induced phosphorylation. Simi-
Although NOX inhibitors should generally relieve oxida- larly, we can assume that DPI-mediated ROS production is
tive stress, several lines of evidence for APO and DPI responsible for the observed effects on Akt and Erk activation
exist demonstrating the opposite. It was reported that DPI which is in contrast to the response to APO. Properties of
inhibits pentose phosphate pathway (PPP) responsible for the general antioxidant NAC in sense of attenuation of kinase
synthesis of NADPH, a redox cofactor important for many phosphorylation were more profound during treatment in
antioxidant enzymes, thus making the cells more prone to complete medium. Notably, we did not observe changes of
oxidative stress [18]. Suggested mechanism included direct Stat3 phosphorylation in presence of any drugs tested in our
inhibition of NADP-dependent enzymes such as glucose 6- study. The most striking effect was detected downstream of
phosphate dehydrogenase, glyceraldehyde 3-phosphate dehy- PI3K on the level of S473 Akt phosphorylation when APO
drogenase, and lactate dehydrogenase. In agreement with treatment was employed. In serum starved cells, in FBS or LIF
our data, evidence from different group exists, showing that activated cells, and also in the presence of complete medium,
DPI is exerting prooxidative effects in given cell types and APO decreased Akt phosphorylation when short term impact
conditions [17]. Further, it was reported that APO, contrary to was studied. It was earlier reported that vanillin and some
DPI, stimulates PPP which is known to be a subsequent step of its derivatives, including APO, readily inhibited PI3K in
following oxidative stress exposure. This was prevented by lung adenocarcinoma cell line [69]. These authors are also
addition of GSH into medium, further suggesting that APO- suggesting, in agreement with our data, that radical scaveng-
induced GSH oxidation might be involved in observed PPP ing or other antioxidant properties of those compounds are
activation [11]. However, in a different study APO opposingly not responsible for the observed effect. Thus, the mode and
increased the synthesis of the GSH through activation of mechanism of inhibition needs to be further clarified.
transcription factor AP-1. Notably, levels of GSSG were not Remarkably, critical role of PI3K was also described for
altered, implying that APO itself does not cause oxidative the process of NOX activation [70]; thus it can be hypoth-
stress [61]. Many studies are highlighting that APO is a esized that some of the APO inhibitory actions towards
prodrug that must be first metabolized through oxidation to NOXs might be also mediated via PI3K inhibition. To
its oligomeric form; thus certain redox environment must further elucidate impact of APO-induced PI3K inhibition,
be present in order to achieve full APO activation [62] nar- we examined the level of homeodomain transcription fac-
rowing its function preferably to cells with strong ROS pro- tor Nanog, which was suggested as a downstream target
duction like stimulated endothelial cells [63] or professional of PI3K and is also recognized as an important intrinsic
phagocytes [64]. In compliance with these findings, it was regulator of stem cell pluripotency [28]. After 24 hours of
demonstrated that APO can act both as an antioxidant and APO treatment, the level of Nanog was dramatically reduced.
as a prooxidant, depending on the cell type and its oxidizing Interestingly Akt phosphorylation was upregulated by APO
potential [9, 10]. This is in agreement with our experiments after 24 hours compared to untreated cells, and this increase
performed on nonphagocytic low-level ROS cells where APO could be reverted by addition of NAC, although Nanog
might rather contribute to oxidative stress as demonstrated levels remained diminished, suggesting that this impairment
by live imaging. Interestingly, different modifications of APO is perhaps not related to APO-induced modulation of ROS
are suggested for in vivo experiments [65], further revealing levels in the cell. Phosphorylation of both Akt and Erk was
the complexity of this issue. augmented by DPI treatment that may be attributed to its
Critical features of mES, pluripotency, self-renewal, and above described prooxidative effect, as it was reduced by NAC
unlimited proliferation, are predominantly exerted through supplementation in the case of Akt.
actions of cytokine LIF, which is routinely added to the Next, we examined the impact of NOX inhibitors on
culture medium. Binding of LIF to its receptor triggers canonical Wnt signaling, as it also plays a distinctive role in
the activity of three major intracellular signaling cascades: regulation of embryonic stem cells [31, 71] and the modulator
JAK/Stat3, PI3K/Akt, and MAPK/Erk. These pathways con- of this pathway, GSK3, is a known PI3K downstream [72].
verge to regulate the gene expression pattern typical for mES GSK3 regulates Wnt pathway by phosphorylating -catenin
[27]. To clarify possible effects of APO and DPI on selected on multiple sites that enhances its subsequent degradation
signaling pathways, the modulation of Stat3, Akt, and Erk [43]. GSK3 is active in resting cells but is readily inhib-
activation status was analyzed in mES cells. Firstly, mES were ited through the PI3K/Akt-mediated phosphorylation of N-
serum and LIF depleted in order to increase cellular response, terminal serine residues (S9 in GSK3 and S21 in GSK3)
as cultivation in complete medium leads to a constant [72]. Therefore, in the conventional view, it is assumed that
activation of those pathways. Stimulation by LIF and FBS activity of PI3K/Akt should promote canonical Wnt signal-
was employed to reactivate signaling [25, 66]. As expected, ing. In contrast to this, experimental evidence argues against
LIF addition induced preferably Stat3 response while FBS, this simplified scenario, as Akt activation failed to promote
containing growth factors and cytokines, augmented Akt and Wnt/-catenin signaling when insulin and constitutively
Erk signaling. To further analyze effect of drugs on selected active Akt were administrated [73]. Moreover, it was reported
kinases, we examined their phosphorylation status also in that Axin-associated GSK3, responsible for mediating -
complete medium after 1-hour and 24-hour treatment, to catenin degradation, represents just a minor fraction of GSK3
elucidate early changes and later cell response in signaling cellular content and this complex is not accessible to Akt
phosphorylation, thus preventing PI3K/Akt/Wnt cross-talk This research is also supported by Collaboration between
[74]. In our experiments, we did not see any effect on Masaryk University and Karolinska Institutet, Project KI-
basal Wnt transcription activity, when NOX inhibitors or LY MU Grant CZ.1.07/2.3.00/20.0180, and the Employment of
was employed. Moreover, treatment of both APO and LY Newly Graduated Doctors of Science for Scientific Excellence
decreased GSK3 S9 phosphorylation, further questioning Program CZ.1.07/2.3.00/30.0009 cofinanced from European
the simplified model of PI3K/Wnt cross-talk mediated solely Social Fund and the state budget of the Czech Republic.
by level of GSK3 inhibition. However, after Wnt3a induction,
both LY and APO augmented Wnt transcription activity.
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http://dx.doi.org/10.1155/2016/5213532
Research Article
Protective Effects of D-Penicillamine on Catecholamine-Induced
Myocardial Injury
Michal kha,1 Pavlna Hakov,2 Jan Martin,3 Tom Filipsk,1

Katelina V^ov,4 Jaroslava Vvrov,5 Magdalena HoleIkov,5 Pavel Homola,2
Libor Vtek,4,6 Vladimr Palicka,5 Tom imRnek,2 and Plemysl Mladjnka1
1
Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague,
Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
2
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague,
3
Department of Pharmacognosy, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague,
4
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague,
Katerinska 1660/32, 121 08 Prague, Czech Republic
5
Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Charles University in Prague,
Sokolska 581, 500 05 Hradec Kralove, Czech Republic
6
4th Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague,
U Nemocnice 2, 128 02 Prague, Czech Republic
Correspondence should be addressed to Premysl Mladenka; mladenkap@faf.cuni.cz
Copyright 2016 Michal Rha et al. This is an open access article distributed under the Creative Commons Attribution License,
Iron and copper release participates in the myocardial injury under ischemic conditions and hence protection might be achieved
by iron chelators. Data on copper chelation are, however, sparse. The effect of the clinically used copper chelator D-penicillamine
in the catecholamine model of acute myocardial injury was tested in cardiomyoblast cell line H9c2 and in Wistar Han rats. D-
Penicillamine had a protective effect against catecholamine-induced injury both in vitro and in vivo. It protected H9c2 cells against
the catecholamine-induced viability loss in a dose-dependent manner. In animals, both intravenous D-penicillamine doses of 11
(low) and 44 mg/kg (high) decreased the mortality caused by s.c. isoprenaline (100 mg/kg) from 36% to 14% and 22%, respectively.
However, whereas the low D-penicillamine dose decreased the release of cardiac troponin T (specific marker of myocardial injury),
the high dose resulted in an increase. Interestingly, the high dose led to a marked elevation in plasma vitamin C. This might be
related to potentiation of oxidative stress, as suggested by additional in vitro experiments with D-penicillamine (iron reduction
and the Fenton reaction). In conclusion, D-penicillamine has protective potential against catecholamine-induced cardiotoxicity;
however the optimal dose selection seems to be crucial for further application.
1. Introduction developed countries. However, in some cases, due to con-

traindications or inaccessibility of adequate medical devices,
Cardiovascular diseases remain the main cause of mortality particularly in developing countries, pharmacotherapy using
in developed countries. The main culprit is atherosclerosis fibrinolytics is still used. Irrespective of the procedure
associated with coronary heart disease which can lead to used, the recovery of blood flow of previously ischemic
its acute form, acute myocardial infarction [1]. The current myocardium is associated with a phenomenon called reperfu-
treatment based mainly on the percutaneous coronary inter- sion paradox [2]. This is based on a burst of reactive oxygen
vention has largely substituted other treatment modalities in species (ROS) generated when previously ischemic tissue is
OH OH
HO
HO
N HO N H NH2
HO H R
R H3 C OH
HS
Catecholamine CH3 O
Aminochrome
-R: -R:
-CH3 = epinephrine -CH3 = adrenochrome Chelator
-CH2 (CH3 )2 = isoprenaline -CH2 (CH3 )2 = isoprenochrome D-penicillamine
(a) (b) (c)
Figure 1: Chemical structures of compounds under investigation: (a) catecholamines epinephrine /(R)-4-(1-hydroxy-2-
(methylamino)ethyl)benzene-1,2-diol/ and ISO /(RS)-4-(1-hydroxy-2-(isopropyl-amino)ethyl)benzene-1,2-diol/; (b) aminochromes,
that is, catecholamine oxidation products, adrenochrome /3-hydroxy-1-methyl-2,3-dihydro-1H-indole-5,6-dione/ and isoprenochrome
/3-hydro-xy-1-isopropyl-2,3-dihydro-1H-indole-5,6-dione/; and (c) copper chelator D-PA /(2S)-2-amino-3-methyl-3-sulfanyl-butanoic
acid/.
reexposed to oxygenated blood flow. Release of free or loosely D-PA can protect cardiomyoblast derived cells and/or modify
bound transition metals, namely, iron and copper, into the the acute myocardial injury caused by administration of
circulation plays an important role in this phenomenon, well synthetic catecholamine isoprenaline (ISO) in rats.
documented in experimental animals [3, 4]. Our research
group has previously shown that iron chelators might protect 2. Materials and Methods
the myocardium from acute catecholamine-induced cardiac
injury, which resembles the acute myocardial infarction in 2.1. In Vitro Experiments
many aspects [5, 6]. Similarly, other groups have demon-
strated some degree of protection by iron chelators in both 2.1.1. Reagents and Solutions. The catecholamines epineph-
experimental in vivo and clinical studies [79]. rine (EPI, Figure 1(a)) and ISO (Figure 1(a)), copper
Much less attention has been paid to copper and its chela- chelator D-PA (Figure 1(c)), ferric chloride hexahydrate
tors and their potential effects in acute myocardial ischemic (FeCl3 6H2 O), ferrous sulphate heptahydrate (FeSO4 7H2 O),
conditions. In human, copper elevation in patients with 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4 ,4 -disulphonic
acute myocardial infarction has been well documented and acid sodium salt (ferrozine), sodium acetate, acetic acid, 4-(2-
a recent study has clearly demonstrated that higher serum hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES),
copper is associated with higher cardiovascular mortality HEPES sodium salt, hydroxylamine, salicylic acid, and
[10, 11]. To our knowledge, only one study tested the effect 2,3-dihydroxybenzoic and 2,5-dihydroxybenzoic acids were
of a known copper chelator in an experimental model of purchased from Sigma-Aldrich (USA). Deferoxamine was
myocardial ischemia. That study documented protective role purchased from Novartis (Switzerland), dimethyl sulfoxide
of neocuproine against hydrogen peroxide induced cardiac (DMSO) was purchased from Avantor Performance Materials
damage and reperfusion arrhythmias in isolated perfused (USA), and methanol for HPLC was purchased from JT
heart [12]. As far as we know, the effect of a copper chelator Baker (USA). The chemicals and solutions used for cellular
in a whole animal model of myocardial injury has never been cultivation (cultivation media, sera, etc.) were purchased
tested. In the present study, we selected D-penicillamine (D- from Sigma-Aldrich or Lonza Group (Switzerland).
PA), a drug with a long history of clinical use in both copper-
based and non-copper based pathologies. It is considered as 2.1.2. Iron Chelation and Reduction Assay. Ferrozine is a
the standard copper chelator and its rapid effect on copper specific indicator which forms a magenta colored complex
excretion in urine is well documented [13, 14]. Although the with ferrous ions. In principle, ferric ions do not react with
drug is a close derivative of endogenous amino acid cysteine, ferrozine. But the methodology can be extended for the
it is known to have side effects (e.g., severe gastrointestinal assessment of total iron chelation after reduction of ferric
disturbances, rash, proteinuria, and hematological adverse ions by a suitable reductant like hydroxylamine. Similarly,
effects) when given in a long-term basis [15]. This is likely iron reduction can be easily assessed. If a tested compound is
not true for its acute administration, where even the dose of able to reduce these ferric ions into ferrous ions, the indicator
1.2 g/kg of D-PA did not produce toxic effects in rats [16]. rapidly forms a complex with them which is thereafter
For a possible clinical use of D-PA relevant to the acute measured spectrophotometrically [17].
myocardial injury, timely limited administration is suggested Stock solutions of ferric ions and ferrous ions were pre-
and therefore such therapy might be without significant pared in distilled water (Milli-Q RG, Merck Millipore, USA).
adverse reactions. The main aim of this study was to assess if The corresponding fresh working solutions (0.25 mM) were
prepared by dilution with distilled water. Hydroxylamine CA

hydrochloride and ferrozine were dissolved in distilled water; (a) CA: Experiment
D-PA was dissolved in DMSO. Experiments were performed CA
in 15 mM buffers, acetate (pH 4.5 and 5.5) and HEPES (pH 6.8 (b) CA and D-PA: Experiment
and 7.5). Metal chelation experiments were performed in 96- D-PA
well microplates, at least in duplicates, at room temperature CA
using a Synergy HT Multi-Detection Microplate Reader (c) oxCA: Preoxidation Experiment
(BioTek Instruments, Inc., USA) as described previously
[17]. Briefly, for this assessment of iron chelation, various CA
(d) oxCA then D-PA: Preoxidation Experiment
concentrations of D-PA were mixed in mentioned buffers
with ferrous or ferric ions (at a final concentration of 25 M) D-PA
for 2 min. In case of the assessment of total iron chelation, CA
(e) oxCA with D-PA: Preoxidation Experiment
hydroxylamine was then added for reduction of nonchelated
D-PA
iron. The absorbance was measured immediately after the
addition of ferrozine and after 5 min at 562 nm. 24 hours 24 hours
For the determination of the degree of ferric ions reduc- preincubation cellular exposure
tion, various concentrations of the tested compounds were Figure 2: Overview of the protocols for cell experiments using
mixed for 2 min with ferric ions in acetate or HEPES buffers. the H9c2 cardiomyoblast cell line. Catecholamines (CA-ISO or
Afterwards, ferrozine was added and absorbance was mea- epinephrine) were added to H9c2 cells either as freshly prepared
sured at 562 nm immediately and after 5 min. Hydroxylamine solutions (a and b) or after 24 h preincubation in the cell-culture
was used as a positive control (100% reduction). medium at 37 C (oxidized oxCA) (ce). D-PA was added either
at the beginning of 24 h cellular experiments (b and d) or at the
start of 24 h catecholamine preincubation before the 24 h cellular
2.1.3. Inhibition of Iron-Catalyzed Production of Hydroxyl Rad- experiments (e).
icals. Ferrous ions react with hydrogen peroxide to produce
hydroxyl radical (the Fenton reaction). The formed radical
can be trapped by salicylic acid and its ensuing products
(2,3-dihydroxybenzoic and 2,5-dihydroxybenzoic acids) can to mimic the situation in postmitotic cardiomyocytes [20].
be detected by HPLC [18]. Briefly, ferrous ions were mixed Pyruvate was omitted because it is an antioxidant and
with the tested compounds dissolved in methanol in different may interfere with ROS-related toxicity. The lipophilic com-
concentration ratios for 2 min. Salicylic acid and hydrogen pounds were dissolved in DMSO, yielding a final concentra-
peroxide (both 7 mM) were added subsequently, and the tion of 0.1% in all experimental groups. At this concentration,
mixture was then analysed by HPLC (Dionex Ultimate 3000, DMSO had no effect on cellular viability.
Dionex Corp., USA) with Eclipse Plus C18 column (4.6
100 mm, 3.5 m, Agilent Inc., USA), using 40% methanol 2.1.5. Cell Experiments. All cell experiments were based
and 0.085% aqueous solution of phosphoric acid as a mobile on the ability of catecholamines to undergo spontaneous
phase. All experiments were checked by addition of internal oxidation to a number of chemically related products [21].
standard, that is, known amounts of 2,3-dihydroxybenzoic For that reason, there were created five different protocols
and 2,5-dihydroxybenzoic acids. for the study of toxic and protective properties of compounds
under investigation (Figure 2). In brief, work solutions of cat-
2.1.4. Cell Culture. The H9c2 cell line derived from embry- echolamine (ISO or EPI) used in all experiments were either
onic BD1X rat heart tissue [19] was obtained from the freshly prepared or 24 h preoxidized (i.e., left spontaneously
American Type Culture Collection (ATCC, USA). Cells were to oxidize) at 37 C. The copper chelator D-PA was added into
cultured in Dulbeccos modified Eagles medium (DMEM; the solution with catecholamine either at the beginning of the
Lonza) supplemented with 10% heat-inactivated fetal bovine cell experiment itself or at the beginning of 24 h preoxidaton
serum (FBS; Lonza), 1% penicillin/streptomycin solution of catecholamine.
(Lonza), and 10 mM HEPES buffer (pH 7.4; Sigma). Cell
cultivation was performed in 75 cm2 tissue culture flasks from 2.1.6. Neutral Red Uptake Assay for Assessment of Compounds
Techno Plastic Products AG (TPP) at 37 C in a humidified Cytotoxicity. Cellular viability was determined using the
atmosphere of 5% CO2 in air. Cells were subcultured twice assay based on the ability of viable cells to incorporate neutral
in a week when they reached approximately 90% confluence red (NR; Sigma). This well-established assay consists in the
(i.e., every 3rd-4th day). readily penetration of intact cell membranes by weak cationic
For particular experiments, cells were seeded into appro- dye, NR, and its accumulation in the lysosomes of viable
priate microplates (TPP) at given cellular density. The cells [22]. H9c2 cells seeded in 96-well plates at a density of
medium was changed for serum-free cell-culture medium 10,000 cells per well were incubated with compounds under
(pyruvate-free DMEM (Sigma) supplemented with 1% peni- investigation (alone or in combinations) for 24 h. Half of
cillin/streptomycin solution (Lonza) and 10 mM HEPES the medium volume from each well was removed at the
buffer (pH 7.4; Sigma)) 24 h prior to all cellular experiments. end of incubation, and the same volume of medium with
Serum deprivation was used to stop cellular proliferation NR was added, yielding a final concentration of 40 g/mL.
After 3 h at 37 C, the supernatant was discarded, and the (positive control), D-PA11+ISO, and D-PA44+ISO, while the
cells were fixed in 0.5% formaldehyde supplemented with others are designated as C (negative control), D-PA11, and D-
1% CaCl2 for 15 min. The cells were then washed twice with PA44.
phosphate buffered saline and solubilized with 1% acetic acid
in 50% ethanol for 30 min of continuous agitation; thus the 2.2.3. Anaesthesia and Surgery. The animals had free access
accumulated NR was released into the extracellular fluid. The to water and diet during the first 12 h after drug adminis-
light absorption (optical density) of released NR was mea- tration. The rats were then fasted for the next 12 h before
sured using a microplate spectrophotometer Tecan Infinite the surgery. Animals were anaesthetized with i.p. injection
200 M (Tecan, Switzerland) at = 540 nm. The viability of containing aqueous solution of urethane (Sigma-Aldrich)
experimental groups was expressed as a percentage of the in a dose of 1.2 g/kg. Surgical and instrumental procedures
untreated control (100%). were similar to our previous studies [6]. Briefly, the left
common iliac artery was connected to a pressure transducer
2.1.7. Epifluorescence Microscopy for Imaging of Cellular MLT0380/D (ADInstruments, Australia) via a polyethylene
Morphology Changes. Changes in cellular morphology were catheter (0.5/1.0 mm filled with heparinized saline 50 IU/mL)
observed and imaged using an inverted epifluorescence for arterial blood pressure measurement. A high-fidelity
microscope Nikon Eclipse TS100 with 1040x air objec- pressure-volume micromanometer catheter (Millar pressure-
tives (Nikon, Japan) equipped with a digital camera 1300Q volume catheter SPR-838 2 F, 4E, 9 mm, Millar Instru-
(VDS Vosskuhler GmbH, Germany) and the software NIS- ments Inc., USA) was inserted into the left heart ventricle
Elements AR 3.0 (Laboratory Imaging s.r.o., Czech Republic). through the right common carotid artery. Both pressure
The cellular viability was visualized using nuclei staining transducer and Millar pressure-volume catheter together
with Hoechst 33342 (Molecular Probes, USA) and propidium with subcutaneous electrodes for the ECG standard limb lead
iodide (PI; Molecular Probes), which are well-established II MLA1215 (ADInstruments) were connected to PowerLab
and sensitive probes to determine apoptosis and necrosis: with LabChart 7 software (ADInstruments). Data were ana-
Hoechst 33342 is a blue-fluorescent probe ( ex = 360 nm, em lyzed for 30 min, and necessary calibrations with hypertonic
= 460 nm) staining all nuclei. In apoptotic cells, chromatin saline (2 20 L of 25% w/w sodium chloride solution) were
condensation occurs and apoptotic cells can thus be iden- performed at the end of the experiment. A blood sample
tified as those with condensed and more intensely stained was collected from the abdominal aorta into a heparinized
chromatin. The red-fluorescent ( ex = 560 nm, em = 630 nm) test tube (170 IU). Following the experiment, all surviving
DNA-binding dye, PI, is unable to cross the plasma mem- animals were killed painlessly in anaesthesia by intravenous
brane of living cells but readily enters necrotic (or late-stage administration of 1 mL of 1 M aqueous solution of potassium
apoptotic) cells and stains their nuclei red. H9c2 cells seeded chloride (Sigma-Aldrich).
in 12-well plates at a density of 75,000 cells per well were
incubated with compounds under investigation (alone or in 2.2.4. Biochemical Analyses. Cardiac troponin T (cTnT) was
combinations) for 24 h. After that, cells were stained with measured in serum; vitamin C and vitamin E were measured
10 g/mL Hoechst 33342 and 1 g/mL PI for 10 min at 37 C. in plasma. cTnT was determined by high sensitive electro-
chemiluminescence immunoassay (Cobas e411, Roche) using
2.2. In Vivo Experiments two monoclonal antibodies specifically directed against cTnT.
Vitamin E was measured with fluorometric detection after
2.2.1. Animals. Forty-six Wistar Han male rats were obtained deproteinization in an HPLC system LC-10A (Shimadzu,
from Meditox (Czech Republic) and housed in cages located Japan). Analogously, vitamin C was measured after depro-
in a special air-conditioned room with a periodic light-dark teinization by electrophoresis using UV detection (PrinCE
(12-12 h) cycle for 2 weeks. During this period, they were 750, Netherlands).
provided with free access to tap water and standard pellet For lipid peroxidation measurement, 100150 mg of heart
diet for rodents. After the acclimatization period, healthy rats tissue was diluted 1 : 9 (by weight) in ice-cold 0.1 M potassium
weighing approximately 390 g were used for the experiments. phosphate buffer. The tissue was diced and then sonicated
The study was approved by the Experimental Animal with ultrasonic cell disruptor (Model XL2000, Misonics,
Welfare Committee of Charles University in Prague, Faculty USA). 20 L of heart sonicate was incubated for 30 min
of Pharmacy in Hradec Kralove, and conformed to the Guide in 37 C with 100 M ascorbate (Sigma-Aldrich) and 6 M
for the Care and Use of Laboratory Animals published by the FeSO4 as previously described [23]. The amount of CO
US National Institutes of Health (NIH Publication Number produced into vial was quantified by gas chromatography
85-23, revised 1996). with reduction gas detector serving as an index of lipid
peroxidation.
2.2.2. Experimental Design. The rats were randomly divided
into six groups. Firstly, D-PA in the doses of 11 or 44 mg/kg or 2.3. Data Analysis. The amount of nonchelated or reduced
water for injection (B. Braun, Germany, 2 mL/kg) was admin- iron was calculated from the difference of absorbance
istered into the tail vein. ISO (Sigma-Aldrich, 100 mg/kg, between the tested sample (with ferrozine) and its corre-
50 mg/mL) or water for injection was given s.c. 5 min later. sponding blank (without ferrozine) divided by the difference
The groups in which rats received ISO are designated as ISO of the control sample (the known amount of iron without
100 ##
##
100

#
[% of control]

Cell viability
75
% of chelated Fen+
75 #
#
50
50
#
25

0
25
+ + + + + + + + + + + + + + +
10
30
100
300
1000
10
30
100
300
1000
10
30
100
300
1000
10
30
100
300
1000
0
Concentration CA (300 M) oxCA (60 M) oxCA (60 M)
1:10 1:1 10 : 1 100 : 1 of D-PA (M) and D-PA then D-PA with D-PA
Ratio D-PA : Fen+
Alone +EPI
pH 4.5 pH 4.5 (total iron)
+ISO
pH 5.5 DMSO (Fe2+ )
pH 6.8 DMSO (total iron) Figure 4: Cell viability studies using the H9c2 cardiomyoblast
pH 7.5 cell line. Figure shows dose-dependent own toxicity of D-PA (10
Figure 3: Iron-chelating activity of D-PA at (patho)physiologically 1000 M, left) and its protective effects against toxicity induced
relevant pH conditions and nonbuffered conditions (DMSO). by catecholamines (CA) ISO and EPI. D-PA was added to freshly
prepared catecholamine solution before the start of 24 h cell exper-
iments (CA and D-PA, middle left); D-PA was added immediately
before cellular experiments to catecholamines preincubated for 24 h
the tested substance) and its control blank. The concentration in cell-culture medium (oxCA then D-PA, middle right); or D-PA
of hydroxyl radical was calculated as the mean of the was preincubated for 24 h together with catecholamines and then
samples mixed (1) with methanol and (2) with added internal added to cells (oxCA with D-PA, right). Schematic overview of
standards. Calculations in animal study were performed as experimental protocols is in Figure 2. Cell viability was determined
by neutral red uptake assay and expressed as a percentage of the
previously described [6].
untreated control group. Data are presented as means SD; = 4;
Grubbs test was used for detection of outlier values in ani- statistical significance (ANOVA) < 0.05 versus control and # <
mal and cell culture studies. Data were expressed as mean 0.05 versus corresponding catecholamine group.
SD. For multiple comparisons, one-way ANOVA followed
by Tukeys multiple comparisons test (in vivo experiments)
and Bonferroni post hoc analysis (cell culture study) were
used. Differences between groups were considered significant protection was observed especially in cells treated with
at < 0.05 unless indicated otherwise. All statistical preoxidated catecholamines. In this case, 300 and 1000 M
analyses were performed by GraphPad Prism version 6.0 of D-PA completely prevented cell death.
for Windows (GraphPad Software, USA) except for the cell Changes in cell morphology were observed and imaged
culture experiments where the statistical software SigmaStat using an inverted epifluorescence microscope in H9c2 car-
for Windows 3.5 (Systat Software, Inc., USA) was used. diomyoblasts (Figure S1 in Supplementary Material avail-
able online at http://dx.doi.org/10.1155/2015/5213532). While
necrotic (or late-stage apoptotic) cells were found after 24 h
3. Results preincubated catecholamines ISO and EPI (ox-ISO and ox-
3.1. In Vitro Experiments. Since metal chelators are generally EPI, resp.), 300 M D-PA markedly prevented the damage
not very specific which seems to be true for D-PA as well of cardiomyoblasts. D-PA alone did not apparently change
[24, 25], we were firstly interested if D-PA can chelate iron. cellular morphology.
Our competitive spectrophotometric approach confirmed
the ability of D-PA to chelate iron. However, the chelating 3.2. In Vivo Study. With regard to the protective action of
capacity dropped with decreasing pH (Figure 3). D-PA in vitro, its in vivo effects on rats were consecutively
As we have previously shown, compounds with iron- tested. The s.c. ISO dose of 100 mg/kg (after i.v. dose of
chelating potential could reduce catecholamine cardiotox- solvent) caused 4 deaths of 11 rats (36% mortality) within
icity both in vitro and in vivo [5]; we firstly assessed the 24 h. Intravenous premedication by two doses of D-PA (11 and
effect of D-PA on cell damage caused by catecholamines EPI 44 mg/kg) decreased the mortality in ISO-treated animals in
and ISO and their oxidation products (oxEPI and oxISO) both doses (Figure 5(a)). The lower dose of D-PA was more
in cardiomyoblast H9c2 cell line (Figure 4). D-PA alone beneficial in comparison with the higher one (14% versus
did not significantly influence the cell viability in the tested 22%, 1 death of 7 rats and 2 deaths of 9 rats, resp.). Four
concentration range (101000 M) which is achievable in rats (1 in the ISO group, 2 in D-PA44+ISO, and 1 in D-PA44
plasma by administration of D-PA [26]. However, D-PA was group) died during the surgery. These deaths, however, were
able to restore the viability after catecholamine treatment likely not to be linked to the type of treatment but were
in a dose-dependent manner. Interestingly, D-PA mediated caused by problems during the surgical procedure. Except for
P < 0.01
40 150
P < 0.001
30
100
(g/L)
(%)
20
50
10
0 0
Control ISO D-PA11 D-PA44 D-PA11 D-PA44 Control ISO D-PA11 D-PA44 D-PA11 D-PA44
+ ISO + ISO + ISO + ISO
(a) (b)
P < 0.01
0.4
5 P < 0.05

0.3 4

3
(mV)
0.2
(%)
0.1
1
0.0 0
Control ISO D-PA11 D-PA44 D-PA11 D-PA44 Control ISO D-PA11 D-PA44 D-PA11 D-PA44
+ ISO + ISO + ISO + ISO
(c) (d)
P < 0.01
150 P < 0.001
100
(mol/L)
50
0
Control ISO D-PA11 D-PA44 D-PA11 D-PA44
+ ISO + ISO
(e)
Figure 5: Mortality (a), changes in levels of cardiac troponin T (b), QRS-T junction (c), wet ventricles weight index (d), and levels of vitamin
C in plasma (e). Statistical significance versus control: < 0.05, < 0.01, and < 0.001.
one animal in D-PA44 group, mentioned above, no deaths

occurred in any control rat which did not receive ISO. 100
The lower mortality in the group with the lower D-
PA dose in comparison to positive ISO group apparently
% of reduced Fe3+
75
corresponded to a lower release of cTnT (Figure 5(b)). There
was not a significant difference in cTnT levels between D-
PA11+ISO and ISO. However, there was also no difference 50
between D-PA11+ISO and controls suggesting a partial pro-
tection by this lower dose. Unexpectedly, higher dose of D-
PA with ISO increased significantly cTnT when compared to 25
both the controls and the ISO group. Only very low serum
levels of cTnT were found in all rats which did not receive ISO.
0
Data on QRS-T junction (analogous to ST-segment elevation 1:10 1:1 10 : 1 100 : 1
in human) corresponded to cTnT results (Figure 5(c)). An
Ratio D-PA : Fe3+
increase of wet ventricles weight index after administration of
ISO indicates myocardial oedema in these acute experiments pH 4.5 pH 7.5
pH 5.5 DMSO
[27]. The effects of both lower and higher dose of D-PA on pH 6.8
wet ventricles weight index (Figure 5(d)) were opposite to our
above-mentioned results. The higher dose of D-PA improved Figure 6: Reduction of ferric ions by D-PA at four pH conditions
myocardial oedema, while the lower dose only tended to and at nonbuffered conditions (DMSO).
improve it.
Hemodynamic Parameters. ISO treatment did not signifi-

cantly modify blood pressure, but it accelerated heart rate and Since iron reduction may lead to potentiation of the
decreased stroke volume and ejection fraction 24 h after drug Fenton chemistry, we also tested the influence of D-PA on
administration. D-PA alone in both doses did not affect the the in vitro production of hydroxyl radical in the iron-
mentioned parameters in comparison to the solvent control catalyzed Fenton reaction using the HPLC system (Figure 7).
group. Coadministration of ISO with D-PA did not lead In contrast to the standard iron chelator deferoxamine (DEF)
to the normalization of the hemodynamic parameters with which dose-dependently decreased the formation of hydroxyl
exception of the higher dose, where the drop in stroke volume radical, D-PA showed more complex, inverse bell-shaped
was prevented. Representative hemodynamic parameters are behaviour. At very low ratios of D-PA to Fe2+ , D-PA efficiently
depicted in Figure S2. blocked the Fenton reaction, while at higher ratios the effect
Nonsignificant changes were observed in other hemo- of this amine was neutral.
dynamic parameters, especially left-ventricular end-diastolic
pressure, developed pressure, /max , and /min . The 4. Discussion
relaxation parameter (the time constant of left ventricular iso-
volumic pressure decay, tau) was likely due to high variability D-PA is a drug with complex mechanism of action as
only insignificantly elevated in the ISO group in comparison can be documented by its large therapeutic indication. D-
to controls (data not shown). PA represents the basic decoppering agent used for the
treatment of Wilsons disease [28]. Other indications include
Markers of Oxidative Stress. The level of vitamin C in plasma especially rheumatoid arthritis, cystinuria, scleroderma, or
in both D-PA groups and solvent were almost identical heavy metal poisoning [29]. Our first idea was to test
and only insignificant drop was observed in ISO group this drug mainly because of its known effect on copper
which was analogous in the case of D-PA11+ISO group. homeostasis. Although its copper chelation effect may be
However, the higher dose of D-PA markedly increased serum limited [14, 30], it is well known for its ability to induce copper
concentration of vitamin C in ISO-treated rats (Figure 5(e)). excretion in the urine [13, 26]. Therefore, the main hypothesis
No changes in serum concentration of vitamin E and lipid was that copper released from ischemic myocardium could
peroxidation in the heart were observed (data not shown). be chelated or mobilized to urine by D-PA, which might
protect myocardium from catecholamine-induced injury.
3.3. Additional In Vitro Experiments. Since the results of Furthermore, since iron is also known to be released during
lower and higher dose of D-PA were divergent in the in vivo myocardial ischemia [3, 4] and metal chelators including
study and, in particular, the higher dose of D-PA markedly D-PA are generally not completely selective, we also eval-
increased levels of vitamin C in plasma, additional in vitro uated D-PA chelating effect on iron ions. Interestingly, D-
experiments were performed in order to assess possible anti- PA showed pronounced and stable iron chelation effects at
or prooxidant action of D-PA. D-PA substantially reduced neutral or slightly acidic pH in our experiments (Figure 3).
ferric ions and this reduction was dependent on the acidity While more than 80% of ferrous ions were chelated at the
of the environment, especially at pH 4.5 or at nonbuffered concentration ratio of 10 : 1 (D-PA : Fe2+ ) at slightly acidic
conditions; iron was completely reduced from the ratio 2 : 1 conditions or physiological pH, neither cuprous nor cupric
(D-PA : Fe3+ , Figure 6). ions were bound by D-PA at the same ratio [30]. This was
rather unexpected since a previous report has shown that 75

the affinity of D-PA for iron is not particularly high and
lower than that for other metals [31]. However, chelation 50
% of OH formation
and/or increased excretion of both iron and copper might 25
be positive for the use of D-PA in the case of ROS-mediated
myocardial injury. Moreover, before we started to test D-PA 0
in rats, we firstly tested its effects on cardiomyoblast cells.
D-PA had apparently very low toxicity and clear protection 25
against catecholamines was seen at this level (Figures 4 and
S1). Our previous experiments with a strong and lipophilic 50
iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) 75
revealed that the protective action of chelation with SIH was
associated with at least two distinct effects: (i) slowing down 1 : 10000 1 : 1000 1 : 100 1 : 10 1:1
the progressive catecholamine oxidation to reactive toxic Ratio compound : Fe2+
intermediates and (ii) reduction of toxicity of the already DEF
formed oxidation products [21]. The present results suggest D-PA
that D-PA may act by similar mechanisms. Figure 7: The impact of D-PA and the standard iron chelator
The injury caused by synthetic catecholamine ISO resem- deferoxamine (DEF) on hydroxyl radical formation. Grey area
bles in many aspects the acute myocardial infarction in represents the error of the method.
human. For an in vivo model of myocardial infarction, we
administered ISO in a dose of 100 mg/kg s.c. which is a
generally used dosage to produce significant mortality with
about 1/3 of deceased animals within 24 h [5, 6, 32, 33]. from D-PA after the addition of copper was documented
The mortality in our current study was analogous (36%). [34, 35], (2) D-PA in high doses produces intracellular
In agreement with in vitro findings, both doses of D-PA oxidative stress in cell experiments but antioxidant enzymes
reduced the mortality but the effect was not dose-dependent might be both upregulated or downregulated [36], and (3) a
(lower dose decreased the mortality to 14% while the higher recent clinical study showed that treatment with D-PA led to
to 22%). In our previous experiments, only two iron- significant decrease in the activity of glutathione peroxidase,
chelating compounds had clearly protecting effects on the one of the most important antioxidant enzymes in blood, and
mentioned model in the terms of the most severe parameter, a tendency to increase the total antioxidant capacity [37]. The
the mortality: 2-pyridylcarboxaldehyde 2-thiophenecarboxyl latter is a similar finding to the significant plasma vitamin C
hydrazone in the dose of 20 mg/kg completely prevented the increase reported in the present study. On the other hand,
mortality, while the lower dose of 10 mg/kg did not have it should be mentioned that, in healthy animals, D-PA in
an effect [5] and dexrazoxane in the dose of 20.4 mg/kg these acute settings was very well tolerated in both doses
reduced the mortality to 12.5% [6]. Other drugs with iron- and we did not observe any negative haemodynamic changes.
chelating properties, namely, deferoxamine and lactoferrin, Therefore, as suggested above, the negative effect of the higher
were without any effect, while rutin deteriorated the mortality dose of D-PA seems to be related to ischemia caused by
[5, 32, 33]. administration of ISO or to direct effect of this catecholamine
The results with D-PA were different since both lower and and/or its oxidation products. Ischemia leads to a decrease
higher doses of D-PA evoked apparently diverse effects on our in pH [38] and one of the plausible mechanisms of D-PA
model. The lower dose, 11 mg/kg, is equimolar to 50 mg/kg prooxidation is iron reduction seen particularly at lower
of deferoxamine and was selected for reason of comparison pH (Figure 6). In addition, copper reduction by D-PA was
on the molar basis with other chelators as in our previous documented in our previous study [30]. It is well known that
studies [5]. Moreover, this dose fits in the dosage range in reduction of iron or copper intensifies the Fenton reaction
the treatment of Wilsons disease [28]. Higher dose was added due to redox-cycling of the catalyst [39] and therefore D-
to the study protocol because of partially protective effects of PA could increase the production of hydroxyl radical. D-
the lower dose. Both doses decreased the mortality, but the PA behaviour in relation to the Fenton chemistry appears to
mechanism does not seem to be identical. The lower dose be similar to some reducing antioxidants [40], since D-PA
decreased the release of cTnT suggesting cardioprotection, acted as an efficient antioxidant in low concentrations but
while the higher dose had rather an opposite effect on cTnT its protective properties decreased or were even reversed at
but decreased the wet ventricles weight index and normalized higher ratios (Figure 7). This may explain the unexpected
the stroke volume. From the biochemical point of view, it effect of the higher dose of D-PA in the animal model but
is quite surprising that the higher dose of D-PA evoked we are aware that it is not possible to directly transform
the marked increase in plasma vitamin C (Figure 5(e)). It in vitro data into in vivo situation. Although it is tempting
is not easy to explain this result which was found only in to speculate that increased oxidative stress could explain
the combination of D-PA with isoprenaline but not with the why the higher dose was not more efficient than the lower
solvent. It is therefore likely linked to the toxic effects of one, more in vivo experiments will be needed to confirm
isoprenaline. Oxidative stress might play a role. We are not it. On the other hand, the high dose of D-PA had some
the first to suggest it, since (1) hydrogen peroxide production protective effects which were very different from those of
the lower dose and might be caused by other mechanisms [3] E. Berenshtein, B. Vaisman, C. Goldberg-Langerman, N.
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http://dx.doi.org/10.1155/2016/9268531
Research Article
A New Method to Simultaneously Quantify the Antioxidants:
Carotenes, Xanthophylls, and Vitamin A in Human Plasma
Mariel Colmn-Martnez,1 Miriam Martnez-Hulamo,1,2 Esther Miralles,3

Ramn Estruch,2,4 and Rosa M. Lamuela-Ravents1,2
1
Nutrition and Food Science Department, XaRTA, INSA, Pharmacy School, University of Barcelona, Avenue Joan XXII s/n,
08028 Barcelona, Spain
2
CIBER CB06/03 Fisiopatologa de la Obesidad y la Nutricion, CIBEROBN, Choupana s/n, 15706 Santiago de Compostela, Spain
3
Scientific and Technical Services, University of Barcelona, Baldiri Reixac, 10, 08028 Barcelona, Spain
4
Department of Internal Medicine, Hospital Clnic, IDIBAPS, University of Barcelona, Villarroel, 170, 08036 Barcelona, Spain
Correspondence should be addressed to Rosa M. Lamuela-Raventos; lamuela@ub.edu
Received 22 June 2015; Revised 19 August 2015; Accepted 24 August 2015
Copyright 2016 Mariel Colman-Martnez et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD)
method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is
presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl
ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z--carotene isomers, and 1 fat-soluble
vitamin (retinol) were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and
330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight
volunteers. Results. Limits of detection were between 0.1 g/mL for lycopene and astaxanthin and 1.3 g/mL for 15-Z--carotene.
Recoveries were ranged between 89% and 113% for -carotene and astaxanthin, respectively. Accuracy was between 90.7% and
112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three
lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy,
precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies.
1. Introduction Carotenoids comprise a group of fat soluble phytochem-

icals widely distributed in nature, responsible for the colours
Several epidemiologic studies have shown that oxidative of many fruits and vegetables, as well as certain animal tissues
stress plays an essential role in the pathogenesis of many and leaf coloration after the degradation of chlorophyll [13].
degenerative diseases, such as cancer, diabetes, age-related Considering the number of double bonds in the molecules,
eye diseases, and cardiovascular diseases [16] and it has carotenoids can be found with cis or trans configuration (or
been suggested that antioxidants may exert a protective role E/Z isomers). In general the all-E form is predominant in
against these chronic diseases by defending against oxidative nature but numerous researches show that more than 50% of
damage [7, 8]. There is an increasing interest in the analysis some carotenoids, as lycopene, present in human plasma and
of carotenoids and some fat-soluble vitamins such as retinol tissues are Z-isomers [14] and it is believed that geometrical
(vitamin A) due to their antioxidant properties and their rela- configuration of carotenoids could have implications in the
tionship with the prevention of chronic diseases [912]. The solubility, absorption, and transport in humans [15, 16] or
characterization and quantification of carotenoids and retinol even geometrical isomers of provitamin A carotenoids have
in human plasma are essential for best interpretation of epi- different vitamin A activities [17]. The enhanced absorption
demiologic studies linking oxidative stress, diet, and health. of lycopene Z-isomers is hypothesised to result from higher
solubility in mixed micelles, the shorter length of the Z- nonpolar layer was removed and the remaining aqueous
isomers, and/or a lower tendency to aggregate [14, 18]. This plasma mixture was reextracted as described above. The two
hypothesis was supported by studies in both animals and nonpolar extracts were combined in a glass vial and dried
humans [18, 19]. under nitrogen gas at <25 C followed by a reconstitution with
Among fat-soluble vitamins, retinol exerts an important 300 L of MTBE. Then, the samples were stored into insert-
antioxidant action via the inhibition of lipid peroxidation and amber vials for HPLC at 20 C until the day of analysis.
has free-radical-scavenging properties [11, 20]; meanwhile,
carotenoids are known to be effective quenchers of singlet 2.1.4. Instrumentation. Chromatographic analysis was car-
oxygen, as well as strong scavengers of different reactive ried out in an HP 1100 HPLC system (Hewlett-Packard, Wald-
oxygen species (ROS) [21]. bronn, Germany), consisting of a quaternary pump and an
High-performance liquid chromatography (HPLC) is autosampler coupled to a diode array detector DAD G1315B.
one of the most used techniques for the identification of Chromatographic separation was performed on a reversed-
carotenoids [22, 23] as well as vitamin A in human plasma phase column YMC Carotenoid S-5 m, 250 mm 4.6 mm
[2426]. The advantages of the use of this technique are (Waters, Milford, MA), maintained at 25 C, and connected to
speed, sensitivity, and accuracy for the determination of a precolumn YMC Guard Cartridge Carotenoid 20 4.0 mm
the compounds in addition to the economy of the solvents i.d, S-5 m. The guard column was replaced every 500 injec-
required and the simple coupling with other techniques. tions. The integration was performed with Agilent Chem-
In light of the importance of these compounds for health Station Software. The DAD detector was adjusted at 450 nm
maintenance, an accurate determination and quantification for the carotenoids and 330 nm for retinol, respectively. All
in plasma is necessary. The aim of this study is to develop a compounds were identified by retention time compared with
simple HPLC method to identify the main carotenoids and pure standards as well as by the UV-Vis spectra of each
their geometrical isomers as well as retinol, one of the major compound.
antioxidant fat-soluble vitamins, in human plasma.
2.1.5. Chromatographic Conditions. The chromatographic
2. Experimental Procedures separation was performed using the following solvents: Milli-
Q water (A), methanol (B), and MTBE (C). Solvent A was
2.1. Materials and Methods used isocratically at 4% while the following linear gradient
was used for B ( (min), %B): (0.0, 90); (40.0, 40); (60.0,
2.1.1. Reagents and Standards. Carotenoids and vitamins 6); (62.0, 90); (72.0, 90). Twenty microliter aliquots of the
standards: retinol, astaxanthin, lutein, zeaxanthin, E--apo- samples were injected in the HPLC-DAD system. Total run
8 -carotenal, cryptoxanthin, 15-Z--carotene, 13-Z--caro- time was 72 minutes at a flow rate of 0.6 mL/min.
tene, -carotene, -carotene, 9-Z--carotene, and lycopene
were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.1.6. Quality Parameters. The method was fully validated
Methanol (MeOH) and methyl-tert-butyl ether (MTBE) based on the criteria of the AOAC International and the
of HPLC grade were obtained from Panreac Quimica SA U.S. Department of Health and Human Services Food and
(Barcelona, Spain). Ultrapure water (Milli-Q) was generated Drug Administration (FDA) [28, 29]. The quality parameters
by a Millipore System (Bedford, MA, USA). Human plasma established for the validation of the method were accuracy,
and butylated hydroxytoluene (BHT) were acquired from intra- and interday precision, recovery, limit of detection
Sigma-Aldrich. (LoD), limit of quantification (LoQ), and linearity.
Three dilutions were prepared from the stock solution in
2.1.2. Preparation of Standard and Stock Solutions. Individual MTBE to the final concentrations of low (1.5 g/mL), medium
working standards of retinol, astaxanthin, lutein, zeaxanthin, (4 g/mL), and high (9 g/mL) for all analytes for precision
E--apo-8 -carotenal, cryptoxanthin, 15-Z--carotene, 13- and accuracy assays.
Z--carotene, -carotene, -carotene, 9-Z--carotene, and Accuracy consisted in the closeness of agreement
lycopene were prepared at a concentration of 1 mg/mL between the measured value and the reference value and was
in MTBE. All working standards were manipulated under established by repetitively spiking blank plasma with three
protection of light to minimise light-induced isomerisation, known concentrations of analyte standards: low, medium,
stored in eppendorf tubes, and kept at 20 C until use. and high with respect to the calibration curves, in five
The stock solution used to spike plasma samples was replicates. The results were determined as the percentage of
prepared by mixing individual working standards at a con- the ratio of the mean observed concentration and the known
centration of 50 mg/mL in MTBE. spiked concentration in the biological matrices. The mean
value should be within 15% of the nominal value.
2.1.3. Extraction and Isolation of Carotenoids and Retinol. Intraday precision and interday precision were consid-
Plasma was subjected to a liquid-liquid extraction procedure ered using five determinations per three concentration levels:
previously described by our working group [27]. Briefly, low, medium, and high in a single analytical run or in three
800 L of plasma was mixed with 800 L of ethanol and 2 mL different days, respectively. The precision of the method
of hexane/BHT (100 mg/L). Then, 300 L of stock solution was assessed on the % RSD (percentage of relative standard
was added followed by a vortex-mixing for 1 minute and deviation) of intra- and interday repeatability and the values
centrifuged at 2062 gr for 5 minutes at 4 C. The upper determined at each concentration level should not exceed
15% of RSD, according to the regulation of the AOAC and Wilcoxon test for paired comparisons. Statistical significance
FDA. was set at < 0.05.
Recovery was accomplished by preparing seven-point
calibration curves and seven-point external curves, spiked 3. Results and Discussion
before and after extraction, respectively. The detector
response obtained from the amount of analyte added to and 3.1. HPLC Method Development
extracted from the biological matrix was compared to the
detector response obtained for the same concentration of 3.1.1. Extraction. For the extraction of carotenoids and
the pure authentic standard. A linear regression between retinol, precipitation of proteins from plasma as a prior step
the ratio analyte concentration against the calculated is required. To achieve this issue, most methods use organic
concentration has been applied, and the slope multiplied by solvents such as methanol, ethanol, or acetonitrile [30].
100 corresponded to the analyte recovery. Some studies use ethanol-BHT at different concentrations
Limit of detection (LoD) and limit of quantification [31, 32] while others use ethanol-ascorbic acid [33] or ethanol-
(LoQ) were determined by comparing measured signals from saline [34] for protection of carotenoids besides deproteiniza-
samples with the low concentration of analyte with those of tion. Different solvents and solvent combinations have been
blank samples and establishing the minimum concentration described in the literature for removing lipophilic analytes
at which the analyte can be reliably detected or quantified. A from biosamples. Hexane alone [35, 36] or combined with
signal-to-noise ratio in the order of 3 : 1 and 10 : 1, for LoD and other solvents, such as hexane/acetone [37], hexane/ether
LoQ, respectively, was considered acceptable. [34], or hexane/ethanol/acetone/toluene [38], seems to be
Linearity was tested by assessing signal responses of the most used solvents for the extraction of carotenoids and
target analytes from plasma samples spiked in duplicate at lipophilic compounds from plasma. There are few studies
seven different concentrations and by calculation of linear where hexane-BHT is used for protection of carotenoids dur-
regression. ing the extraction [39, 40] while other authors prefer to use
hexane-saline [34]. Both mixtures, hexane-BHT and hexane-
saline, were tested for extraction. Recoveries obtained with
2.2. Method Application: Pilot Dietary Intervention Study hexane-saline were between 57.4% and 86.9% correspond-
ing to E-lycopene and 13-Z--carotene, respectively, versus
2.2.1. Biological Material. To assess the efficiency in the
recoveries range between 73.4% and 90.4% corresponding
identification of carotenoids and retinol in human plasma,
to E-lycopene and -carotene, respectively, using hexane-
the present method was applied to a small-scale prospective,
BHT as extraction solvent. Due to its simplicity, speed,
open and controlled, single-arm intervention study con-
and good recoveries achieved, ethanol for deproteinization
ducted in eight volunteers free of cardiovascular disease but
and repeated extraction with hexane-BHT were chosen for
with high risk of developing it, aged 69.9 3.8 years with a
performing this study.
mean body mass index of 32.3 3.8 kg/m2 .
The volunteers were instructed to avoid the consumption
3.1.2. Separation and Identification. Numerous HPLC
of tomato and tomato-based products 3 days before the study.
methods are reported for separation and identification of
On the experimental day, after 12 hours of fasting, blood
carotenoids and fat-soluble vitamins from diverse complex
samples were collected early in the morning to quantify
matrices such as food, food products, or plasma. The use of
carotenoids and retinol as baseline. After that, all volunteers
isocratic or gradient systems coupled to different types of
have followed a similar diet developed by a trained dietitian,
columns and/or coupled to different detectors or even the
which took into account their preferences and tastes, as well
use of different temperatures for the stability of the analytes,
as the consideration that participants were diabetic, obese,
depending of the compounds studied is the best described
hypertensive, and/or dyslipidemic. Each day during 4 weeks,
technique for these purposes.
participants consumed 250 mL of tomato juice before dinner
Reversed-phase HPLC with C18 columns and isocratic or
and at the end of the study blood samples were taken for
gradient elution seems to be the modality most commonly
comparing with baseline. All samples were stored at 80 C
used for identification and quantification of carotenoids and
until analysis.
fat-soluble vitamins [4144]. One of the major problems in
The study protocol was approved by the Ethics Commit-
the identification of carotenoids and lipophilic compounds
tee of Clinical Investigation of the University of Barcelona
using C18 columns seems to be the separation of geometrical
(Spain), and the clinical trial was registered at the Inter-
isomers of carotenoids [4147]. Tzeng et al. [33] have devel-
national Standard Randomized Controlled Trial Number
oped an isocratic method for the identification of carotenoids
(ISRCTN20409295). Written informed consent was obtained
using a C18 column, but they could only separate three
from all participants.
carotenoids: lutein, lycopene, and -carotene. Olmedilla et al.
[40] also have described a gradient method with a C18 column
2.3. Statistical Analysis. Statistical analysis was performed but could not identify isomers of the carotenoids. To solve this
using the SPSS Statistical Analysis System (version 22.0; SPSS issue, polymeric C30 columns were developed for separation
Inc, Chicago, IL). Data are presented as means and stan- of Z-E isomers [22, 48, 49]. The use of this kind of column has
dard deviation (SD). Statistical differences between the two allowed us the separation of the twelve compounds studied,
interventions were analysed by the nonparametric statistical including 3 Z-isomers of -carotene and another 3 Z-isomers
0,35
Trans--apo-8 -Carotenal
13-Z--Carotene
0,09
Cryptoxanthin
Retinol
0,08 0,30
0,07
0,06
-Carotene -Carotene
0,25
9-Z--Carotene
0,05
0,04
15-Z--Carotene
0,03 0,20
0,02
Astaxanthin
E-lycopene
Zeaxanthin
0,01 0,15
(AU)
Lutein
(AU)
0,00
0,01 0,10
0,02
0,03 0,05
0,04
0,05 0,00
0,06
0,07 0,05
0,08
0,09
5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 5 0 5 10 15 20 25 30 35 40 45 50 55 60 65
(a) DAD Sig = 330 nm (b) DAD Sig = 450 nm
Figure 1: Representative HPLC chromatogram of carotenoids and retinol standards in MTBE.
0,035 0,035
9-Z--Carotene
Zeaxanthin
13-Z--Carotene
Retinol
15-Z--Carotene
13-Z-Lycopene
E--apo-8 -Carotenal
E-Lycopene
0,030 0,030
Cryptoxanthin
-Carotene
9-Z-Lycopene
5-Z-Lycopene
Astaxanthin
0,025 0,025
-Carotene
0,020 0,020
Lutein
0,015 0,015
0,010 0,010
0,005 0,005
0,000 0,000
(AU)
(AU)
0,005 0,005
0,010 0,010
0,015 0,015
0,020 0,020
0,025 0,025
0,030 0,030
0,035 0,035
0,040 0,040
0 5 10 15 20 25 30 35 40 45 50 55 60 65 0 5 10 15 20 25 30 35 40 45 50 55 60 65
(a) DAD Sig = 330 nm (b) DAD Sig = 450 nm
Figure 2: Representative HPLC chromatogram of carotenoids and retinol in human plasma corresponding to a volunteer after tomato juice
intervention.
of lycopene. Figures 1 and 2 show a representative HPLC- and fat-soluble vitamins from complex matrixes such as food
DAD chromatogram of carotenoids and retinol in a standard samples, human plasma, and human adipose tissue using a
mixture and a human plasma sample, respectively. single C30 column kept at 35 C. All these techniques allow
Likewise lutein and zeaxanthin are reported as two the separation and identification of lipid compounds such as
difficult compounds to separate on monomeric C18 columns carotenoids and fat soluble vitamins from different matrices
[42, 43]. Gueguen et al. [44] found an inadequate resolution but are complex and require coupling of columns and use of
of lutein and zeaxanthin with the method proposed using a temperature or combination of detectors, which is not always
C18 column and an isocratic elution system; Thibeault et al. possible in all laboratories.
[31] were not able to differentiate lutein and zeaxanthin with In this study, carotenoids and retinol were separated in
these same conditions. A high resolution can be achieved by a single run on a reversed-phase column using a gradient
using polymeric C30 columns with gradient elution [22, 49, system of water, methanol, and MTBE. The mobile phase
50]. With the present method, all analytes, including lutein was optimized in order to obtain the best separation of the
and zeaxanthin, have reached a resolution higher than 1.5, compounds in the shortest time possible and to achieve this,
indicating a good separation of the compounds and a good several gradients were assessed. The best results obtained
symmetry of all peaks. Results are shown in Table 1. for the conditions were described in the chromatographic
Coupling of photodiode array detector (DAD) and fluo- conditions section.
rescence detector (FLD) is the technique chosen by Epler et
al. [51] and Liu et al. [32] for identification of carotenoids and 3.2. Validation Parameters
retinols from human serum and foods. Using more complex
techniques, such as photoisomerization of some analytes as a 3.2.1. Linearity. According to the maximal reported value
prior step, Ferruzzi et al. [49] identified 13 lycopene isomers in plasma for each analyte, plasma samples were spiked in
(Z/E) by using an electrochemical detector (ECD). Lyan duplicate at seven different concentrations ranged from LoQ
et al. [46] and Lee et al. [47, 52] have identified various of each compound to 10 g/mL. The analytical procedure was
carotenoids by the coupling of two different detectors or linear over the concentration range tested with the correlation
columns. Other authors like Gleize et al. [53] have set up a coefficient from 0.9952 to 0.9984 for all compounds in plasma
gradient method for the identification of eleven carotenoids samples, demonstrating a good linearity of the curves. Table 4
Table 1: Resolution of the analytes studied. Table 2: Accuracy and recovery of the compounds studied.
Analyte Rt Wavelength Width Analyte Accuracy (%) Recoveries (%)

Resolution
(min) (nm) (min) Retinol 105 9 96 3
Retinol 8.54 330 0.6 N.d. Astaxanthin 99 7 113 6
Astaxanthin 34.07 450 0.38 2.3 Lutein 99 13 112 9
Lutein 36.55 450 0.72 1.4
Zeaxanthin 101 11 107 5
Zeaxanthin 38.24 450 0.53 2.2
E--apo-8 -carotenal 98 10 94 3
E--apo-8 -carotenal 40.01 450 0.29 7.7
Cryptoxanthin 103 12 96 3
Cryptoxanthin 43.94 450 0.22 6.9
15-Z--carotene 90 13 101 2
15-Z--carotene 46.96 450 0.22 1.4
13-Z--carotene 105 12 92 5
13-Z--carotene 47.57 450 0.21 1.3
-carotene 98 12 89 4
-carotene 48.12 450 0.2 2.9
-carotene 97 13 96 2
-carotene 49.57 450 0.31 2.3
9-Z--carotene 112 7 93 3
9-Z--carotene 50.76 450 0.2 33.0
E-lycopene 100 12 91 3
E-lycopene 64.29 450 0.21 33.0
N.d.: not determined.
= 2[(Rt) (Rt)]/( + ).
= resolution. lycopene; 0.2 g/mL for retinol, zeaxanthin, E--apo-8 -
Rt = retention time.
= width. carotenal, cryptoxanthin, -carotene, and 9-Z--carotene;
0.4 g/mL for lutein and 13-Z--carotene; 0.5 g/mL for -
carotene, and 1.3 g/mL for 15-Z--carotene. The LoQ was
summarizes the correlation coefficients of the curves of all from 0.3 g/mL to 4.4 g/mL for astaxanthin and 15-Z--
compounds. carotene, respectively. All LoD and LoQ values obtained are
expressed in Table 4. In a study carried out by Mitrowska et
3.2.2. Accuracy and Precision. Accuracy and intra and inter- al. [55] similar values were obtained for E--apo-8 -carotenal,
day precision were studied. All compounds analysed met astaxanthin, and lycopene. In another study developed by
the acceptance criteria to not overcome 15% RSD in both Talwar et al. [43] a similar value for retinol but not for the
intra- and interday precision and in the three concentration other compounds studied can be observed; nonetheless, with
levels. The highest values were 15% belonging to astaxanthin, the presented method we have achieved the separation and
zeaxanthin, -carotene, -carotene, and 13-Z--carotene. identification of 11 carotenoids and 1 fat-soluble vitamin in a
Accuracy results obtained were between 90.7% and 112.2% single run.
being within limits of accuracy, 85115%. The method pro-
posed demonstrated good accuracy and precision in plasma 3.2.5. Plasma Levels of Carotenoids and Retinol. The method
samples, asserting that was feasible for the determination was used for measuring concentrations of carotenoids and
of carotenoids and retinol in human plasma. Results are retinol in human plasma samples. All compounds were
expressed in Tables 2 and 3. determined following the procedure described above and the
results are expressed in Table 5.
3.2.3. Recovery. Recovery for retinol was 96%, similar to Nine of the 12 validated analytes were identified in plasma
the value achieved by Kandar et al. [54]. For carotenoids, at baseline: retinol, astaxanthin, lutein, E--apo-8 -carotenal,
the recoveries were between 89% and 113%, corresponding cryptoxanthin, 13-Z--carotene, -carotene, -carotene, and
to -carotene and astaxanthin, respectively. Comparing our lycopene. It is important to highlight that, besides the iden-
results with those reported by Talwar et al. [43] we achieved tification of the validated compounds, we also have searched
a recovery 18% higher for lutein and 9% higher for - for cis isomers of lycopene which have been suggested to be
carotene with the described method; comparing with the data more bioavailable than E-lycopene, typically found in raw
presented by Tzeng et al. [33], we achieved a better recovery food [18]. In fact, in human plasma, total lycopene is an
for lutein (20% higher), and comparing with Rajendran et al. isomeric mixture containing 40% to 50% as Z-isomers [5]
[22] we obtained a better recovery for lutein (19% higher), and according to the antioxidant properties of lycopene, from
zeaxanthin (13% higher), and cryptoxanthin (7% higher). greatest to least are 5-Z, 9-Z, 7-Z, 13-Z, 15-Z, 11-Z, and all-E
Karppi et al. [39] have reported similar values of recovery lycopene [56]. We have identified 5-Z, 9-Z, and 13-Z lycopene,
except for lutein, zeaxanthin, and -carotene that were 10%, all present in human plasma according to the study carried
17%, and 16%, respectively, lower than in the present study. out by Arranz et al. [27]
The extraction procedure was really effective, since high After tomato juice intervention, an increase in retinol,
recoveries can be observed in Table 2. astaxanthin, cryptoxanthin, 13-Z--carotene, -carotene, 13-
Z-lycopene, 9-Z-lycopene, E-lycopene, and 5-Z-lycopene was
3.2.4. Limit of Detection (LoD) and Limit of Quantification observed, with values between 5.194 and 0.140 g/mL cor-
(LoQ). The LoD found was 0.1 g/mL for astaxanthin and responding to E-lycopene and 13-Z--carotene, respectively.
6
Table 3: Intra- and interday precision.

Precision
1,5 g/mL ( = 5) 6 g/mL ( = 5) 9 g/mL ( = 5)
Analyte Day 1 Day 2 Day 3 RSD Day 1 Day 2 Day 3 RSD Day 1 Day 2 Day 3 RSD
(RSD %) (RSD %) (RSD %) Interday (RSD %) (RSD %) (RSD %) Interday (RSD %) (RSD %) (RSD %) Interday
Retinol 3 4 5 4.9 10 12 8 14.1 13 10 11 14.3
Astaxanthin 7 3 3 14.5 11 13 12 14.6 15 12 12 14.7
Lutein 8 9 4 14.2 12 14 8 14.3 10 9 12 13.3
Zeaxanthin 4 15 4 10.8 12 3 7 12.9 11 11 10 14.8
E--apo-8 -carotenal 11 10 4 14.3 11 12 8 14.7 13 9 12 13.8
Cryptoxanthin 5 13 3 12.1 11 6 9 14.3 11 10 8 13.9
15-Z--carotene 5 13 1 9.4 11 5 9 15.1 10 7 11 13.4
13-Z--carotene 6 14 3 14.9 12 15 12 14.0 11 10 10 14.9
-carotene 4 15 2 9.4 11 15 11 13.7 11 9 10 14.8
-carotene 11 10 8 11.2 11 15 14 14.9 12 6 10 12.8
9-Z--carotene 6 10 7 10.4 12 12 4 13.9 11 11 11 14.8
E-lycopene 5 5 2 11.6 10 13 9 13.8 10 15 8 14.8
RSD: relative standard deviation.
Table 4: Limit of detection (LoD), limit of quantification (LoQ), range of concentration, calibration curve, and correlation coefficient of the
analytes in blank plasma spiked with standard solution.
Analytes LoD (g/mL) LoQ (g/mL) Linearity range (g/mL) Calibration curve Correlation coefficient ()
Retinol 0.2 0.7 0.710 = 69.684 15.857 0.9952
Astaxanthin 0.1 0.3 0.310 = 49.138 + 3.0197 0.9964
Lutein 0.4 1.3 1.310 = 119.37 2.263 0.9954
Zeaxanthin 0.2 0.7 0.710 = 69.14 + 1.53 0.9955
Apo-8 -carotenal 0.2 0.7 0.710 = 249.4 20.908 0.9957
Cryptoxanthin 0.2 0.7 0.710 = 200.48 15.759 0.9952
15-Z--carotene 1.3 4.3 4.310 = 91.235 10.848 0.9984
13-Z--carotene 0.4 1.3 1.310 = 90.591 + 8.7376 0.9969
-carotene 0.5 1.6 1.610 = 243.07 37.135 0.9966
-carotene 0.2 0.7 0.710 = 105.8 2.9437 0.9983
9-Z--carotene 0.2 0.7 0.710 = 75.685 8.4189 0.9959
E-lycopene 0.1 0.3 0.310 = 34.532 4.2861 0.9952
Table 5: Carotenoids and retinol in human plasma before (baseline) and after the dietary intervention.
Concentration
Analytes Baseline After intervention Baseline After intervention
Mean SD (g/mL) Mean SD (g/mL) Mean SD (mol/L) Mean SD (mol/L)
Retinol 1.82 0.37 1.90 0.49 3.46 0.70 3.62 0.93
Astaxanthin 0.76 0.44 0.88 0.37 1.27 0.74 1.48 0.63
Lutein 0.07 0.02 0.06 0.06 0.13 0.03 0.11 0.11
Zeaxanthin N.d. N.d. N.d. N.d.
E--apo-8 -carotenal 0.57 0.03 0.57 0.03 1.38 0.06 1.38 0.08
Cryptoxanthin 0.18 0.12 0.20 0.07 0.32 0.22 0.37 0.12
15-Z--carotene N.d. N.d. N.d. N.d.
13-Z--carotene 0.13 0.00 0.14 0.02 0.23 0.00 0.26 0.04
-carotene 0.22 0.00 N.d. 0.41 0.00 N.d.
-carotene 0.95 0.50 1.09 0.53 1.77 0.93 2.03 0.98
9-Z--carotene N.d. N.d. N.d. N.d.
13-Z-lycopene N.d.a 2.79 1.44a N.d.a 5.20 2.69a
9-Z-lycopene N.d.a 0.38 1.42a N.d.a 0.71 2.64a
E-lycopene 1.15 0.83a 5.19 2.35 a
2.14 1.54a 9.67 4.38a
5-Z-lycopene 0.75 1.10a 3.07 1.43 a
1.41 2.06a 5.72 2.67a
SD: standard deviation.
a
Values in a row with the same letter are significantly different ( < 0.05). Data analyzed by Wilcoxon test for repeated measures.
Among these compounds, lycopene and its isomers have pre- obtained with the present method are higher than those
sented a significant increase after tomato juice consumption reported by Liu et al. [32] who have found a mean of
( < 0.05). Results are shown in Table 5. 0.55 g/mL and 0.56 g/mL in plasma of women and men,
In a study carried out by Pellegrini et al. [57], they found respectively, participants of the Toronto Nutrigenomics and
a concentration of 0.31 g/mL of lycopene and 0.17 g/mL of Health Study.
-carotene in human plasma after a consumption of tomato
puree. In another study performed by Porrini et al. [58], 4. Conclusions
they have seen values in the order of 0.18, 0.21, 0.02, 0.13,
0.03, and 0.23 g/mL for lycopene, lutein, zeaxanthin, - Based on previous reported work, the present method is
cryptoxanthin, -carotene, and -carotene, respectively, after simple, accurate, reliable, sensitive, and selective for the
a daily supplementation of tomato puree. Gartner et al. [59] determination of the antioxidants carotenoids and retinol in
have determined values of 0.02, 0.0002, and 0.001 g/mL for human plasma samples. With this method, all analytes of
lycopene, -carotene, and -carotene, respectively, after a interest were successfully resolved, including lutein, zeax-
consumption of tomato paste. Regarding retinol, the results anthin, and Z-isomers of -carotene which have previously
been reported as critical compounds to identify and separate. micronutrients and pancreatic cancer risk, International Jour-
The sample preparation procedure in this method provides nal of Cancer, vol. 136, no. 6, pp. E665E676, 2015.
excellent recoveries for all analytes. [7] M. R. McCall and B. Frei, Can antioxidant vitamins materially
A total of 8 carotenoids, 3 Z-isomers of the -carotene, 1 reduce oxidative damage in humans? Free Radical Biology and
fat-soluble vitamin, and also 3 Z-isomers of the lycopene were Medicine, vol. 26, no. 7-8, pp. 10341053, 1999.
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mination of their absorption coefficients are needed for their carotenoids and their antioxidant and anti-inflammatory effects
accurate quantification. in cardiovascular care, Mediators of Inflammation, vol. 2013,
Article ID 782137, 11 pages, 2013.
The HPLC method was completely validated and due to
the good results obtained after the intake of tomato juice, [11] J.-M. Yuan, Y.-T. Gao, C.-N. Ong, R. K. Ross, and M. C. Yu,
Prediagnostic level of serum retinol in relation to reduced risk
this method may be applied to evaluate the liposoluble
of hepatocellular carcinoma, Journal of the National Cancer
antioxidants carotenoids and vitamin A in clinical interven- Institute, vol. 98, no. 7, pp. 482490, 2006.
tion antioxidants trials and epidemiological studies status
[12] K. F. Gey, P. Ducimetiere, A. Evans et al., Low plasma retinol
investigations. predicts coronary events in healthy middle-aged men: the
PRIME Study, Atherosclerosis, vol. 208, no. 1, pp. 270274, 2010.
Conflict of Interests [13] W. Stahl and H. Sies, Bioactivity and protective effects of
natural carotenoids, Biochimica et Biophysica ActaMolecular
The authors declare that there is no conflict of interests
Basis of Disease, vol. 1740, no. 2, pp. 101107, 2005.
regarding the publication of this paper.
[14] T. W. Boileau, A. C. Boileau, and J. W. Erdman Jr., Bioavail-
ability of all-trans and cis-isomers of lycopene, Experimental
Acknowledgments Biology and Medicine, vol. 227, no. 10, pp. 914919, 2002.
[15] G. Britton, Structure and properties of carotenoids in relation
This work was supported by CICYT (AGL2013-49083-C3- to function, The FASEB Journal, vol. 9, no. 15, pp. 15511558,
1-R) and the Instituto de Salud Carlos III, ISCIII (CIBER- 1995.
obn) from the Ministerio de Economa y Competitividad [16] A. Schieber and R. Carle, Occurrence of carotenoid cis-isomers
(MEC) and Generalitat de Catalunya (GC) 2014 SGR 773. in food: technological, analytical, and nutritional implications,
Mariel Colman-Martnez would like to thank the University, Trends in Food Science and Technology, vol. 16, no. 9, pp. 416
Research and Information Society Department of the Gen- 422, 2005.
eralitat de Catalunya (FI-DGR 2013) and Miriam Martnez- [17] K. J. Scott and D. Rodriquez-Amaya, Pro-vitamin A carotenoid
Huelamo would like to thank the MICINN predoctoral conversion factors: retinol equivalentsfact or fiction? Food
program. Chemistry, vol. 69, no. 2, pp. 125127, 2000.
[18] W. Stahl and H. Sies, Uptake of lycopene and its geometrical
isomers is greater from heat-processed than from unprocessed
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http://dx.doi.org/10.1155/2016/4985063
Research Article
Relation between Endothelial Nitric Oxide Synthase Genotypes
and Oxidative Stress Markers in Larynx Cancer
K. Yanar,1 U. akatay,1 S. AydJn,1 A. Verim,2 P. Atukeren,1

N. E. zkan,3 K. Karatoprak,4 T. Cebe,4 S. Turan,3 E. Ozkk,5
G. Korkmaz,3 C. CacJna,3 O. Kkhseyin,3 and E. YaylJm3
1
Department of Medical Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University, 34098 Istanbul, Turkey
2
Department of Otorhinolaryngology/Head and Neck Surgery, Haydarpasa Numune Educational and Research Hospital,
34668 Istanbul, Turkey
3
Department of Molecular Medicine, Institute of Experimental Medicine, Istanbul University, 34393 Istanbul, Turkey
4
Cerrahpasa Faculty of Medicine, Istanbul University, 34098 Istanbul, Turkey
5
Department of Neuroscience, Institute of Experimental Medicine, Istanbul University, 34393 Istanbul, Turkey
Correspondence should be addressed to I. Yaylm; ilhanyaylim@gmail.com
Received 11 June 2015; Revised 10 August 2015; Accepted 11 August 2015
Copyright 2016 K. Yanar et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nitric oxide synthase (eNOS/NOS3) is responsible for the endothelial synthesis of nitric oxide (NO ). G894T polymorphism leads
to the amino acid substitution from Glu298Asp that causes lower NOS3 activity and basal NO production in NOS3 894T (298Asp)
allele carriers compared with the GG homozygotes. NO acts as an antioxidant protecting against Fentons reaction which generates
highly reactive hydroxyl radicals. Allelic variation of NOS3 may influence an individuals risk of laryngeal cancer (LC). In the
current study we have examined the possible relationship between NOS3 G894T genotypes and various systemic oxidative damage
markers such as protein carbonyl, advanced oxidation protein products, Cu, Zn-superoxide dismutase, thiol group fractions, and
lipid hydroperoxides in LC patients. Genotyping was carried out by PCR-RFLP. In LC patients with TT genotype, Cu, Zn-superoxide
dismutase activities and nonprotein thiol levels were significantly higher than the controls. In patients with GT and GG genotype,
high levels of lipid hydroperoxides showed statistical significance when compared to controls. Our results indicate a potential
relationship among G894T polymorphism of NOS3, and impaired redox homeostasis. Further studies are required to determine
the role of NOS3 gene polymorphism and impaired plasma redox homeostasis.
1. Introduction Thus, oxidative stress markers in LC have been a step-

forwarding issue and have received attention by various
Laryngeal cancer (LC) represents about 30% of the malignant investigators [4, 5].
tumors of head and neck cancers, and this corresponds to Balance of the NO levels has a critical importance in cell
8% of all cancer types worldwide [1]. Similar to other cancer fate. Nitric oxide synthase (NOS) is the main source of the
types, LC is a multifactorial disease which can be induced NO production which produces NO while converting L-
by both genetic and environmental factors [2]. Among the arginine to L-citrulline. Three functional classes of NOS have
various etiological factors sharing in the development of been described so far as endothelial-NOS (eNOS or NOS3),
laryngeal tumors, increased production of reactive oxygen neuronal-NOS (nNOS), and inducible form of NOS (iNOS)
species (ROS) is playing an important role in the development [6, 7]. NOS3 was first defined in vascular endothelial cells;
of an impaired redox homeostasis [3]. In addition, increased however, later studies showed that this isoform can also be
ROS production may increase the mutation rate in a tissue, found in other cell types such as airway epithelia, neurons,
thereby increasing the rate of tumor recurrence. and certain types of tumors [6, 8]. Angiogenesis dependent
tumors have a significant genetic component, including the patients were diagnosed as well-differentiated laryngeal
major causes of morbidity and mortality in LC patients [9]. squamous cell carcinoma by histopathology. This study con-
NO synthesis by endothelial cells plays an important role in sists of 3 women and 55 men with LC and 84 women and
regulating angiogenesis, killing neoplastic cells, and reducing 63 men as healthy controls (HC). There were no significant
tumor cell adhesion to endothelium [10]. differences between patients and HC in terms of age (60.94
Many of the other genetic polymorphisms have been 9.03/56.83 12.38; = 0.062). In addition, smoking status
reported in NOS3 gene [1113], among them G894T poly- and alcohol consumption were significantly different between
morphism (rs1799983), which is located in exon 7 of the groups [ = 0.0001 for both (98.3% (LC)-6.1% (HC)/53.4%
NOS3 gene and leads to the amino acid substitution from (LC)-2.7% (HC), resp.)]. All LC patients and HC had a
Glu298Asp that causes reduced NOS3 activity and basal NO Caucasian ethnic background.
production in NOS3 894T (298Asp) allele carriers compared Some of the patients and control group individuals were
with the GG homozygotes [12, 13]. NO acts as an antioxidant excluded from the study. The HC were selected from the
protecting against Fentons reaction which generates highly volunteers. All LC patients and HC with previous chemother-
reactive hydroxyl (OH ) radicals [14]. apy, radiotherapy, and surgery history were excluded from
It has been proposed that the increased level of oxidized the study. Both LC group and HC group individuals had no
proteins is observed during carcinogenesis and progression known chronic metabolic disease. LC patients and HC with
of the cancer [15, 16]. any vitamin or antioxidant drug supplementation within 12
Reactive OH radicals can cause oxidative damage to var- months before study entry were also not included in current
ious plasma proteins, leading to the loss of enzymatic activity study.
and/or transformation of amino acids into protein carbonyl Fasting venous plasma samples were obtained from LC
groups (PCO) [15, 16]. Protein carbonylation includes reac- patients before operation and healthy case-control individu-
tive aldehydes and ketones formed via different molecular als.
mechanisms: (i) direct oxidation of the polypeptide backbone
by OH radicals leading to truncated peptides; (ii) side 2.2. Isolation of Genomic DNA. For genomic DNA extraction
chains oxidation of lysine, arginine, proline, and threonine; 300 L of whole blood containing EDTA was used. DNA
(iii) reaction of histidine, cysteine, and lysine amino acid samples were isolated according to salting out technique
residues with reactive aldehydes and hydroperoxides (LHP); [22] and quantified by UV spectrophotometry (Biotek US,
for example, PCO are produced by lipid peroxidation; and Winooski, VT, USA). Isolated genomic DNA samples were
(iv) glycation (nonenzymatic glycosylation) of lysine residues stored at +4 C.
forming Amadori rearrangement products (advanced gly-
cated end products: AGE) [17]. The peroxynitrite (ONOO )
is a short-lived reactive nitrogen species that is generated 2.3. PCR Analysis of G894T Polymorphism. Extracted DNA
by the reaction of NO and superoxide (O2 ) radicals was amplified with polymerase chain reaction (PCR). NOS3
[18]. Cellular proteins are oxidized and/or dimerized by Glu298Asp polymorphism was analyzed using primers (for-
peroxynitrite-derived radicals, including thiol groups and ward, 5 AAG GCA GGA GAC AGT GGA TGG A-3 ;
tyrosine residues. AOPPs are PCO and dityrosine-containing reverse, 5 CCC AGT CAA TCC CTT TGG TGC TCA 3 ).
cross-linking protein products and considered a novel marker PCR-restriction fragment-length polymorphism method was
of oxidant mediated protein damage in systemic circulation used for genotyping [23]. PCR products were digested with
[15, 1921]. Advanced oxidation protein products (AOPPs) BanII and then visualized and analyzed with agarose-gel
can be formed during increased oxidative stress by reaction electrophoresis. Genotype analysis was carried out by two
of plasma proteins [19, 20]. Plasma thiols can be classified independent investigators who were unaware of clinical data.
into two major groups: protein thiols and nonprotein thiols. PCR analysis was carried out using BIORADT-100 ther-
Thiol groups can be oxidized by ONOO -derived radicals mal cycler (BIORAD, Hercules, USA). Genomic DNA was
and initiate radical-dependent chain reactions to produce incubated in a total reaction volume of 25 L containing equal
higher oxidation states of sulphur such as sulfinic and sulfonic concentration of the forward primer 5 AAG GCA GGA GAC
acid derivatives [18]. AGT GGA TGG A-3 and reverse primer 5 CC AGT CAA
The aim of the current study was to evaluate the possible TCC CTT TGG TGC TCA 3 (Invitrogen, Carlsbad, Califor-
relation of NOS3 G894T genotypes with the levels various nia, USA), 200 M deoxynucleotide triphosphate, 10x PCR
protein and lipid oxidation markers and antioxidant status buffer pH 8.3 containing MgCl2 15 mM, and 1.5 units of Taq
such as PCO, AOPP, LHP, total thiol protein thiol, nonprotein DNA polymerase (Invitrogen, Carlsbad, California, USA).
thiol, and Cu, Zn-superoxide dismutase (Cu, Zn-SOD) in All genotypes were read by two independent researchers. In
patients with LC. case of any conflicts, the genotype was repeated.
2. Methods 2.4. Assay of Protein Carbonyl Groups. PCO groups were

measured spectrophotometrically with Biotek Synergy
2.1. Subjects. Primary LC patients were enrolled in the H1 Hybrid Multi-Mode Microplate Reader (BioTek US,
current study. Mean duration of the complaints of the LC Winooski, VT, USA).
patients was 4 months. Oropharyngeal, nasal, laryngeal, neck, We analyzed plasma PCO levels as previously described
and systematic examination of all patients were made. All by Reznick and Packer [24] with some of the volumetric
modifications. 2,4-Dinitrophenylhydrazine (DNPH) reagent and interassay variations were 1.3% ( = 8) and 3.4% ( = 9),
reacts with PCO groups to form chromophoric dinitro- respectively.
phenylhydrazones (100 L plasma : 400 L DNPH). DNPH
reagent was prepared in hydrochloric acid. Proteins were pre- 2.7. Assay of Lipid Hydroperoxides. Plasma LHPs concentra-
cipitated with an equal amount of 20% (w/v) trichloroacetic tions were analyzed spectrophotometrically with the method
acid upon the DNPH reaction completed. The resulting pel- of FOX2 (ferrous oxidation with xylenol orange, version 2)
lets were washed three times with 400 L of an ethanol/ethyl [29]. LHPs groups oxidized ferrous ions to ferric ions in
acetate mixture (1 : 1). Washing procedure was performed by dilute acid solution, and the concentration of resultant ferric
mechanical disruption of pellets in ethanol/ethyl acetate mix- ions was determined by using ferric-sensitive dye, which
ture and repelleting by centrifugation at 3000 g for 5 min. was related to the concentration of LHPs. Xylenol orange
Finally, PCO precipitates were dissolved in a 200 L 6 M binds to ferric ions with high selectivity to form a colored
guanidine-HCl solution and the related absorbance values (blue-purple) complex. Fifth microliters aliquots of plasma
were recorded at 360 nm. The molar extinction coefficient of sample were transferred into microcentrifuge reaction vials.
DNPH ( = 22,000 M1 cm1 ) was used for the calculation FOX2 reagent (950 L) was then added, and the samples
of PCO concentration. The intra- and interassay CV% values were mixed on vortex. After incubation with FOX2 reagent
for modified PCO assay were 4.1% ( = 8) and 8.1% ( = 8), at room temperature for 30 min, the final samples were
respectively. centrifuged at 3.000 g at 20 C for 10 min. The resulting
The untreated bovine serum albumin (BSA) and PCO- supernatant fractions were transferred into microplate wells,
bovine serum albumin (BSA) positive control samples were and absorbance values were read at 560 nm against reagent
both prepared according to the method of Lenarczyk et al. blank.
[25] and analyzed with the PCO assay procedure.
2.8. Assay of Cu, Zn-Superoxide Dismutase Activity. This
2.5. Assay of Advanced Oxidation Protein Products. Spec- assay involves the inhibition of nitroblue tetrazolium (NBT)
trophotometric determination of AOPP concentrations was reduction, with xanthine oxidase (XO) used as a super-
determined by modification of Hanasands method [26]. oxide generator. Cu, Zn-SOD activity was determined by
Samples were prepared in the following way: 10 L of plasma, measuring the inhibition rate of substrate hydrolysis in the
40 L of phosphate buffered saline (PBS), and 200 L of citric assay mixture containing 0.3 mmol/L xanthine, 0.6 mmol/L
acid solution (20 mmol/L) were mixed in microplate. One Na2 EDTA, 150 mol/L NBT, 400 mmol/L sodium carbonate,
minute later, 10 L of 1.16 M potassium iodide was added to and 1 g/L BSA. The pH value of the assay mixture needs
the microplate well; the absorbance of the reaction mixture to be adjusted to pH 10.2 [30]. Nine hundred seventy-two
was read at 340 nm against reagent blank. The chloramine- L assay mixture and 13 L XO (167 U/L) were added to
T absorbance at 340 nm is linear within the range of 0 to 25 L plasma. At the end of the 20 min incubation period,
100 mol/liter. AOPP values were given as micromoles per 250 L, 0.8 mmol/L, CuCl2 was added to the well in order
liter of chloramine-T equivalents. The coefficients of intra- to terminate reaction. The final absorbance was read at
and interassay variations were 1.5% ( = 8) and 2.2% ( = 560 nm against reagent blank. Percent inhibition rate was
8), respectively. The untreated BSA and AOPP-BSA positive calculated according to the following equation: blank
control samples were both prepared in vitro and analyzed sample / blank 100. One unit of Cu, Zn-SOD is defined as
according to the AOPP assay protocol [27]. the amount of enzyme needed to exhibit a 50% dismutation
of superoxide radical anion. The coefficients of intra- and
2.6. Assay of Thiol Fractions. Plasma total thiol, nonpro- interassay variations for modified Cu, Zn-SOD assay were
tein thiol, and protein thiol concentrations were deter- 3.2% ( = 8) and 4.5% ( = 8), respectively.
mined by using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB)
as described by Sedlak and Lindsay [28]. We realized some 2.9. Statistical Analyses. Descriptive statistics were expressed
of the modifications for previously described total thiol as mean SD. Statistical analyses were performed by using
method in order to apply small volumes of plasma samples. SPSS (Statistical Package for the Social Sciences) v16.0 soft-
A portion (20 L) of the plasma sample was mixed in 1.5 mL ware. The statistical analyses of the nonnormally distributed
test tube with 400 L of 0.2 M Tris buffer, pH 8.2, and 20 L data of plasma oxidative stress parameters between patients
of 0.01 M DTNB for total thiol group analysis. Nonprotein and controls sharing the same genotypes were performed
thiol samples were assayed in the following way: 20 L of by using Mann-Whitney U test. Genetic frequencies were
plasma was mixed in 400 L of 50% TCA. The test tubes compared in patients and controls by chi-square (2 ) test. A
were vortexed intermittently for 10 min and centrifuged for level of < 0.05 was considered statistically significant.
15 min at 3000 g. Supernatant fractions were assayed as
total thiol. The absorbance values of the resulting samples 3. Results
were read at 412 nm wavelength against reagent blank. The
value of molar extinction coefficient of thiol (-SH) groups at Restriction band pattern of G894T polymorphism in exon 7
wavelength 412 nm is approximately = 13.100 M1 cm1 . of the NOS3 gene is shown in Figure 1.
The PSH group concentrations were calculated by subtracting Characteristics of patients with laryngeal carcinoma are
the nonprotein thiol from total thiol. The coefficients of intra- given in Table 1.
Table 1: Characteristics of patients with laryngeal carcinoma.
Larynx cancer patients

Parameters
= %
Reflux
Yes 26 44.8
No 32 55.2
Family history of any kind of cancer
Yes 17 30.9
No 38 69.1
Tumor location
Glottic 38 69.1
Supraglottic 17 30.9
Tumor grade
T1 8 14.1
T2 10 17.5
Figure 1: Restriction band pattern of G894T polymorphism in T3 30 52.6
exon 7 of the NOS3 gene. Agarose gel electrophoresis of PCR T4 9 15.8
products after endonuclease restriction with enzyme BannII. Lane Lymph node
1, GG homozygote (163 bp and 85 bp); lane 2, GT heterozygote
N0 36 62.1
(248 bp, 163 bp, and 85 bp); and lane 3, TT homozygote (248 bp). M
represents 50 bp ladder. N1 20 34.5
N2 2 3.4
Metastasis
Genotypes and allele frequencies of NOS3 Glu298Asp in Yes 1 1.8
primary larynx cancer patients and their respective controls
No 54 98.2
are shown in Table 2.
Variations in the levels of plasma oxidative stress parame- Differentiation
ters of patients and their healthy controls are given in Table 3. Poor 8 14
The plasma levels of oxidative stress parameters were Medium 38 66.7
determined with manual colorimetric methods according to Well 11 19.3
NOS3 genotypes in LC patients and their respective con- Tumor recurrence
trols. The plasma levels of oxidative stress parameters were
Yes 8 13.8
determined with manual colorimetric methods according to
NOS3 genotypes in LC patients and HC (Table 4). In LC No 50 86.2
patients with TT genotype, plasma nonprotein thiol and Cu,
Zn-SOD levels were significantly higher than those of the Table 2: Genotypes and allele frequencies of NOS3 Glu298Asp in
control group. On the other hand, plasma AOPP levels were primary larynx cancer patients and their controls. Hardy-Weinberg
not significantly different in any genotypes for LC patients equilibrium analysis showed the genotype distribution for the NOS3
and their corresponding HC. In patients with GG and GT gene (G894T) in larynx cancer patients in accordance with Hardy-
genotype, elevated levels of LHP showed statistically high Weinberg equilibrium.
significance when compared to HC. Patients Controls
No significant differences were determined between (%) (%)
genotype and clinical pathologies such as recurrence,
Genotype
lymph node, differentiation, reflux, and also oxidative stress
GG 18 (31) 31 (21.1)
biomarkers. Our results also show that the subjects with
NOS3 homozygote variants may have a risk for reflux 1.74- GT 29 (50) 81 (55.1)
fold compared to heterozygotes. TT 11 (19) 35 (23.8)
= 0.300
Alleles
4. Discussion
G 65 (56.03) 143 (48.64)
It is well known that ROS can initiate oxidative damage in T 51 (43.97) 151 (51.36)
both plasma constituents and cells of systemic circulation = 0.177
such as proteins, lipids, and DNA [14, 15, 31]. Oxidative mod-
ifications of these macromolecules play an important role
in carcinogenesis [1416]. It has been previously concluded favor of lipid peroxidation and oxidative DNA damage in LC
that systemic oxidant/antioxidant balance was impaired in patients [32].
Table 3: Variations in the levels of plasma oxidative stress parameters of larynx cancer patients and their controls.
Patients Controls value

PCO (nmol/mg pr) 1.19 0.08 0.86 0.08 0.008
AOPP (mol/L chloramine-T equivalent) 56.04 5.61 41.04 5.22 0.037
Total thiol (nmol/mg pr) 15.54 1.19 16.23 1.55 0.843
Nonprotein thiol (nmol/mg pr) 3.35 0.30 3.46 0.19 0.703
Protein thiol (nmol/mg pr) 12.17 1.19 12.86 1.51 0.860
LHP (mol/mg pr) 3.71 0.42 1.20 0.12 0.000
Cu, Zn-SOD (U/mg pr) 7.27 0.31 5.99 0.34 0.004
< 0.05; < 0.01.
Table 4: Mean SD values of oxidative stress parameters according to NOS3 genotypes in larynx cancer patients and controls.
GG GT TT
Patients Controls value Patients Controls value Patients Controls value
PCO 1.3 0.2 1.0 0.3 0.517 1.2 0.1 0.9 0.1 0.125 1.1 0.1 0.7 0.1 0.088
AOPP 54.1 9.4 43.4 6.7 0.833 52.7 6.4 34.6 7.8 0.029 66.4 18.9 48.3 10.1 0.562
Total thiol 16.8 3.2 21.9 3.0 0.315 15.5 1.6 13.9 2.0 0.371 14.1 1.2 16.4 3 0.731
Nonprotein thiol 3.4 0.9 4.0 0.5 0.524 2.9 0.3 3.4 0.3 0.363 4.4 0.3 3.3 0.3 0.037
Protein thiol 13.0 3.3 17.7 2.6 0.315 12.8 1.6 10.7 2.1 0.285 9.7 1.3 13.1 2.9 0.628
LHP 4.2 0.8 1.4 0.3 0.006 4.0 0.6 1.3 0.2 0.001 2.4 1.0 1.1 0.2 1.22
Cu, Zn-SOD 6.9 0.4 5.9 0.6 0.279 7.2 1.8 6.4 0.6 <0.05 7.9 0.8 5.5 0.5 0.036
< 0.05; < 0.01; < 0.001.
There are contradictory reports about the possible role It is well known that the extent of the intravascular
of NO in carcinogenesis: some of the studies indicate a oxidative stress is the main risk factor for the occurrence and
potential carcinogenic role of NO related to the promotion progression of various types of cancers [1416, 31]. Neoplastic
of tumor angiogenesis [15, 33]. However, Kong et al. suggest transformations give rise to the generalized oxidative and
the possible protective role of NOS with a potential to reduce nitrosative stress in plasma, and the secondary reactive prod-
the tumor cell adhesion to endothelium [34]. Therefore, ucts of oxidative and nitrosative damage tend to accumulate
being the responsible enzyme of NO production, NOS3 is during the progression of cancer [15, 16]. Plasma nitrite and
thought to be involved in this critical regulation of NO nitrate levels may not be sensitive biomarkers of systemic
synthesis and thus in possible carcinogenic mechanisms NO status, and they reflect not only NO levels but also other
[6]. Almost 400 NOS3 variants have been defined so far reactive nitrogen species in plasma [43, 44]. The accurate
and some of them are known to be related to carcinogenic measurement of the nitrite/nitrate couple is analytically
transformations [35]. A few of these polymorphisms have problematic due to interferences and other methodological
been reported to be significantly associated with the develop- restrictions [44]. Hence, we decided to estimate stable sys-
ment of certain cancer types [3638]. G894T polymorphisms temic oxidative stress parameters in LC patients with G894T
of the NOS3 gene are very important in the angiogenesis polymorphism. Dissimilarities with respect to T allele are
pathway and have also been found to have functional and found when our results are compared to the results of Ritt
clinical significance in malignancies [39, 40]. The basis of et al. [45]. These investigators also found that in patients
our choosing G894T polymorphism among others can be with diabetes who carry the T allele of the G894T the
explained as the product of NOS3 is constitutively expressed magnitude of oxidative stress tends to increase. Reduction in
in endothelial cells and vascular epithelium of the cancer cells the production of NO may be related to increased oxidative
[40, 41]. All these experimental findings suggest that NO stress and the presence of T allele.
may play a significant role in angiogenesis and a prominent Plasma proteins are also the direct target for ROS because
role in human carcinogenesis. The prevalence of this ana- of their high concentrations in systemic circulation. Plasma
lyzed G894T polymorphism in general population represents proteins can be oxidized by a variety of free radicals and
heterogeneity. The source of heterogeneity may arise from oxidants. Oxidative modifications of plasma proteins, such as
many aspects, such as the ethnic region of study, the sample PCO and AOPP, usually result in a loss of protein function.
size, the case and the control group, clinical characteristics Identifying the carbonylation of proteins is critical for the
of different tumors, and the genotyping methodology [42]. determination of intravascular redox homeostasis, and it
Since ethnicity related genetic polymorphism plays an impor- could potentially provide important information concerning
tant role in cancer risk, further studies need to be focused molecular mechanisms underlying the development and
to clarify mortality and morbidity rates for various ethnic progression of cancer linked to oxidative damage [15, 16].
groups. PCOs are early and reliable biomarkers of ongoing protein
oxidation [46]. AOPP can be formed during increased oxida- DTNB: 5,5-Dithiobis(2-nitrobenzoic acid)
tive stress by reaction of plasma proteins such as albumin FOX2: Ferrous oxidation with xylenol orange,
with chlorinated oxidants. Thus, AOPP has been considered version 2
a novel marker of oxidant-mediated protein damage [21]. LC: Laryngeal cancer
AOPP plays an important role in advance phase of oxidative LHP: Lipid hydroperoxides
protein damage, which consists of different types of protein NO : Nitric oxide
oxidation markers such as dityrosine, pentosidine, and PCO NOS: Nitric oxide synthase
[26]. In our study, no statistically significant increase was NOS3: Endothelial-NOS
seen in plasma AOPP levels in patients with TT genotype. PBS: Phosphate buffer saline
No reports are available in current literature that investigates PCO: Protein carbonyl group
G894T polymorphism in exon 7 of the NOS3 gene and PCR-RFLP: PCR-restriction fragment-length
oxidative protein damage in laryngeal cancer. Disruption polymorphism
of redox regulation of plasma proteins may therefore be ROS: Reactive oxygen species.
the result of genotype-related increase in the magnitude of
oxidative stress and occurrence of carcinogenesis.
Nonprotein thiol groups such as glutathione are physi-
Ethical Approval
ological free radical scavengers [28]. Glutathione may be a The ethical protocol of the current research was approved by
primary agent involved in redox regulation of protein thiols. the Haydarpasa Numune Education and Research Hospital,
Plasma Cu, Zn-SOD activity and nonprotein thiol levels were Istanbul, Turkey, Ethics Committee Issue Number: HNEAH-
statistically increased in laryngeal cancer patients with TT KAEK 2013/262.
genotype. The increased levels of aforementioned parameters
may be related to their preventive role for the formation of the
AOPP. On the other hand, plasma Cu, Zn-SOD activities were Consent
not different with G allele and their allele-matched controls.
All subjects provided written informed consent before they
We attribute the increase in antioxidant activity of Cu, Zn-
participated in the study.
SOD and nonprotein thiol groups to a function of effective
homeostatic redox regulation mechanism in patients with
T allele. Phospholipids, cholesterol, cholesterol esters, and Conflict of Interests
triglycerides are major lipids in the plasma. LHPs are the
major primary product of lipid peroxidation and they can be The authors declare that there is no conflict of interests.
measured with FOX2 method [29]. The formation of LHPs
is accepted as an important initial event in the progression Authors Contribution
of lipid peroxidation. The possible pathophysiological role of
increased lipid peroxidation for the aforementioned geno- U. Cakatay, S. Aydn, and I. Yaylm were the principal
types needs to be clarified in future studies. investigators and take primary responsibility for the paper.
A. Verim, S. Turan, and G. Korkmaz recruited the patients.
5. Conclusions K. Yanar, K. Karatoprak, T. Cebe, C. Cacna, and O.
Kucukhuseyin also made contributions to work at laboratory.
Male gender, smoking, and alcohol consumption may induce E. Ozkok and N. E. Ozkan performed the statistical analysis.
laryngeal carcinoma. Since intervention in preclinical con- U. Cakatay, K. Yanar, P. Atukeren, and I. Yaylm wrote the
ditions would have the greatest public health impact, there paper. U. Cakatay, K. Yanar, and P. Atukeren made the
is an important need to pay attention to the dysregulation revisions.
of the redox balance of plasma proteins in high risk groups
of laryngeal cancer. Plasma redox imbalance in patients with Acknowledgment
larynx cancer could be related to the occurrence of risk alleles.
In order to help in early identification of the individuals This work was partially supported in part by a grant funding
harboring high risk for laryngeal cancer, there is also a need to from the Research Fund of the University of Istanbul (UDP-
develop new allele specific and redox-sensitive biomarkers for 34309).
diagnosis. Further studies are required to provide cytological
pattern of the distribution of NOS isoforms and should
compare these results with other systemic oxidative stress
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http://dx.doi.org/10.1155/2016/8214631
Research Article
The Effect of Lycopene Preexposure on UV-B-Irradiated
Human Keratinocytes
Andreia Ascenso,1,2 Tiago Pedrosa,2 Snia Pinho,2 Francisco Pinho,2

Jos Miguel P. Ferreira de Oliveira,2 Helena Cabral Marques,1 Helena Oliveira,2
Sandra Simes,1 and Conceio Santos2
1
Instituto de Investigacao do Medicamento (iMed.ULisboa), Faculdade de Farmacia, Universidade de Lisboa,
Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal
2
Departamento de Biologia, Laboratorio de Biotecnologia e Citomica, CESAM, Universidade de Aveiro,
Campus Universitario de Santiago, 3810-193 Aveiro, Portugal
Correspondence should be addressed to Andreia Ascenso; andreiaascenso@ff.ul.pt
Received 20 May 2015; Revised 3 July 2015; Accepted 6 July 2015
Copyright 2016 Andreia Ascenso et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective
role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h
to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were
analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the
Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene
did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects.
However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared
to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to
nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells
in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our
findings may have implications for the management of skin cancer.
1. Introduction cancer proved to be inadequate in lowering the incidence of

this type of cancer, emphasizing the need to develop novel
Human skin is constantly exposed to the UV irradiation that skin cancer chemopreventive agents. Among the vast number
may induce a number of pathobiological cellular changes. of photochemoprotective agents, botanical antioxidants have
Through lipid peroxidation, protein cross-linking, and DNA given promising results [4]. Two types of chemopreventive
damage, UV-A and UV-B radiation (UVR) can cause pho- agents could be useful for the management of skin cancer.
toaging and photocarcinogenesis [13]. Skin has a variety of Primarily, the agents that could inhibit the damage caused
enzymatic and small molecular antioxidants that can inhibit by UVR may prevent the formation of initiated cells (cells
oxidative damage. However, the excessive ROS production with cancerous potential). Secondly, the agents that could
often exceeds the skin antioxidant ability [4]. In this regard, eliminate the initiated cells may reduce the risk of skin cancer
emphasis on developing novel preventive and therapeutic [5].
strategies based on phytocompounds capable of ameliorating Lycopene is a powerful antioxidant both in vitro and in
the adverse effects of ROS has become an important area of vivo against the oxidation of proteins, lipids, and DNA, and
research. Moreover, primary prevention approaches of skin it has been identified as one of the most potent scavengers of
singlet species of oxygen free radicalsthe highest among the provide a fixation medium for these pigments [25, 26].
carotenoids [6, 7]. At low oxygen tension, it can also scavenge However, the UV-blocking effect of the vehicle is not desired
peroxyl radicals, inhibiting the process of lipid peroxidation in this case, besides the difficulties of using these hydrophobic
[8]. Lycopene was reported as the most quickly depleted systems for cell culture studies.
antioxidant in skin upon exposure to solar radiation [9] and The main limitations of different vehicles used for
might play a role of protection against UVR. Recent research lycopene cell delivery are summarized on Table 1. Each
has been developed to assess if lycopene has potential vehicle provides specific advantages but also offers some
for prevention of skin cancer. In fact, lycopene has been limitations such as cytotoxicity, poor solubility, and crystal-
shown to inhibit proliferation of several types of cancer cells lization in the cell medium [27].
through different mechanisms in in vitro systems [10, 11]. In addition, the half-life of free lycopene in solution at
Chemopreventive antioxidants are mostly studied for their 37 C is less than few hours. Thus, until an efficient method
role as radical scavengers, but this preventive role can be of solubilizing lycopene in aqueous buffers and cell culture
complemented by a corrective activity as selective inducers media is developed, in vitro studies on the effects of lycopene
of apoptosis in transformed cells [12]. Moreover, Ribaya- on living cells will continue to show considerable variation
Mercado et al. [9] suggested a role of lycopene in mitigating between laboratories and cell lines and should be interpreted
photooxidative damage in tissues. with caution [39].
Keratinocytes are the predominant cell type (95%) in the Pfitzner et al. [18] have demonstrated that methyl--CD
epidermis, the outermost layer of the skin [13]. Considering (M--CD) was an improved vehicle for the investigation of
that the principal site of action of UV-B is the epidermis carotenoids and other lipophilic compounds in in vitro test
layer [14], keratinocytes might be more susceptible to UV- systems, compared to organic solvents. Carotenoids-M--
B-induced apoptosis than fibroblasts which are located in CD complex was superior concerning biological availability,
dermis layer (reached by UV-A) [15]. However, keratinocytes missing cytotoxicity and presenting excellent stability when
may be more UV-B resistant in terms of their proliferative compared to other application forms such as organic solvents,
ability as measured by colony survival assays and have greater mixed micelles, liposomes, or beadlets. At least, the solubi-
ability for UV-DNA repair [15]. lization with M--CD was easily and reproducibly achievable
To date, most of the studies on the therapeutic potential under routine laboratory conditions.
of lycopene have been performed in vivo [16, 17]. These According to these literature references [18, 27] and
studies may be obscured by the complexity of biological preformulations studies, we decided to use another similar
system models. In vitro conditions may circumvent some CD derivative, dimethyl--CD (DM--CD), to solubilize and
of these contingencies and complement in vivo data within stabilize lycopene for cell exposure experiments. Depending
the 3Rs perspective (Refine, Replace, and Reduce). Despite on the formulation and exposure conditions, lycopene has
the lower complexity of in vitro systems, the study of cel- been shown to prevent cellular damage or otherwise to sensi-
lular photoprotection by antioxidants could be challenging tize damaged cells leading to increased cell death. We aimed
because of the high chemical instability (especially to air and to study the effects of lycopene as sensitizer and inducer of cell
light) and strong lipophilicity of many antioxidant molecules death in UV-B damaged keratinocytes. Our hypothesis was
such as lycopene. According to Zefferino et al. [11] in vitro that cells preexposed to lycopene would be more sensitized
experiments may occasionally produce inconsistent results to death, in case of subsequent irreversible damage by UV-
due to lycopenes poor solubility in cell culture media [18]. B. For this, the nontumorigenic keratinocyte cell line HaCaT
In fact, lycopene is very hydrophobic (log 15) and is [40] was used. HaCaT cells were preexposed to lycopene for
usually solubilized in organic solvents such as tetrahydrofu- sensitization and subsequently exposed to damaging UV-B
ran (THF). However, an uncontrolled precipitation process irradiation. The effect of lycopene preexposure was analyzed
may occur upon addition to aqueous media, besides the by assays focused on cytotoxicity, genotoxicity, oxidative
high toxicity associated with these solvents. The solubility stress, and apoptosis.
and uptake of these large crystals in the cells are quite
limited and there is almost no protection against chemical 2. Materials and Methods
degradation [19]. Alternative ways of delivering lipid-soluble
compounds include micelles, microemulsions, nanoparti- 2.1. Preparation of Lycopene Complex Solution. In order to
cles, water-dispersible beadlets, artificial liposomes, enriched avoid the use of organic solvents, lycopene (Extrasynthese,
bovine serum, or other formulations, each of which has an Genay, France, with a purity 98%, UV assay) was solubi-
influence on the cellular uptake and compounds stability lized by complexation with dimethyl-beta-cyclodextrin (CD,
[18, 2023]. According to Palozza et al., niosomes provide degree of substitution: 1.8) which was a generous gift from
a suitable, safe, and low-cost vehicle for -carotene in cell Wacker (Stuttgart, Germany). Aqueous solutions of lycopene
culture [24]. Lipid-based delivery systems also show UV- complexed with CD (1 : 4 molar ratio) were prepared under
blocking effects dependent on lipid composition and the aseptic conditions within concentrations of 0, 5, 10, 15, and
particle size (the smaller the particle size, the higher the 20 M from a concentrated lycopene solution previously
sunscreen activity). Lipid matrices can act as sunscreen stirred with CD during approximately 48 h and sonicated
carriers and increase the sun protection factor obtained 30 min (before use), always protected from light and air.
after topical application of UV absorbers (BaSO4 , SrCO3 , Lycopene solutions were always freshly prepared under light
and TiO2 ) incorporated within these carriers because they and air protection and stored at 20 C (except the pure
Table 1: Limitations of different vehicles used for lycopene cell delivery (adapted from Lin et al. [27]).
Vehicle Limitations References

Tetrahydrofuran (THF) Rapid oxidation in media, leading to lycopene instability and cytotoxicity [17, 20, 22, 2832]
Dimethyl sulfoxide (DMSO) Reduced solvent capacity for carotenoids (0.01 mg/mL for lycopene) [20, 3335]
Tween Possible oxidation of carotenoids, after solvent drying and filtration [31]
Micelles Low carotenoid stabilization and increased cytotoxicity [20, 27, 36]
Water-dispersible beadlets Low toxicity, but also low cellular uptake, depends on chemicals that interfere in [29, 37, 38]
assays (e.g., hexane and chloroform)
lycopene standard, stored at 70 C). The osmolarity of the 2.4. Selection of Exposure Conditions
sample containing the highest lycopene concentration was
determined using an automatic osmometer (Knauer, Berlin, 2.4.1. Selection of UV-Irradiation Dose. Ten UV-B lamps
Germany). (Sankyo Denki G8T5E, Kanagawa, Japan) with a peak emis-
When nonspecified, all higher grade reagents were from sion at 312 nm were used as the UV-B source. The UV-
Sigma-Aldrich (St. Louis, MO, USA). All solutions were B irradiation was measured with a VLX 312 radiometer
prepared using ultrapure water obtained in a MILLI-Q equipped with a UV-B sensor (Vilber Lourmat, Marne-la-
System from Millipore (Billerica, MA, USA). Vallee Cedex, France). Around 7,000 cells/well were cultured
in a 96-well cluster plate with complete medium. Twenty-
four hours after, the cells were exposed to five different UV-
2.2. Human Immortalized Keratinocytes (HaCaT) Cell Line. B irradiation doses [based on related literature [4145] of
The HaCaT cell line was obtained from Cell Lines Ser- 75, 150, 200, 225, and 325 mJ/cm2 (moderate-high to very-
vices (CLS) (Eppelheim, Germany). Handling and culture of high dose)]. In order to prevent UV quenching, prior to
these cells were adapted to meet CLS protocol procedures. irradiation, the cell culture medium was replaced by the
Cells were aseptically grown in Dulbeccos modified Eagles same volume of PBS after two washing steps with PBS.
medium (DMEM, no HEPES, no Pyruvate), high glucose, After UV-B irradiation, cells were fed with fresh growth
supplemented with 10% fetal bovine serum (FBS), 2 mM L- medium and incubated for 24 h. Cell metabolic activity was
glutamine, 1% penicillin-streptomycin (10,000 U/mL), and assessed as described below by the MTT assay (often used
1% fungizone (250 U/mL) (Gibco, Life Technologies, Grand as viability method) in order to choose just one UV-B dose.
Island, NY, USA) at 37 C in a humidified atmosphere with Nonirradiated samples were used as negative control.
5% CO2 .
2.4.2. Selection of Complexed Lycopene Dose. From previous
2.3. HaCaT Cell Growth and Confluence under Normal Cul- cell viability results of HaCaT cells exposed to UV-B irradia-
ture Conditions. The standard cell growth conditions were tion, new exposure conditions were tested with different Lyc-
established after the analysis of HaCaT growth curves. HaCaT CD concentrations and a fixed UV-B irradiation dose. Briefly,
cells were seeded in a 6-well cluster plate (300,000 cells/well). cells were seeded and grown in DMEM (Dulbeccos modified
Cell confluence and morphology were daily observed under Eagles medium) supplemented with 10% FBS, 1% penicillin-
microscope. After 3 days, cells were harvested by trypsiniza- streptomycin, and 1% fungizone, for 24 h, and then DMEM
tion according to CLS procedure. Briefly, the culture was medium was replaced with -MEM without nucleosides
rinsed twice with phosphate-buffered saline solution (PBS) (Gibco, Life Technologies) with identical supplementation,
without Ca2+ or Mg2+ (Gibco, Life Technologies, Grand containing complexed lycopene solutions (0, 5, 10, 15, and
Island, NY, USA) and then with PBS containing 0.05% EDTA 20 M). Cells were exposed for 24 h, and after UV-B irra-
to remove desmosomes and incubated at 37 C about 5 diation (225 mJ/cm2 ) HaCaT irradiated cells were incubated
10 min. After this, EDTA solution was replaced by a 1 : 1 under standard culture conditions for a final 24 h period. The
mixture of EDTA/trypsin-solution (final concentrations of metabolic activity of cell culture was analyzed by MTT assay
0.025% and 0.05%, resp.). The trypsinization was achieved in order to choose just one lycopene dose for the subsequent
after incubating at 37 C for 1520 min. At this time, a double analyses (Figure 1).
volume of complete culture medium (with FBS) was added Briefly, for further experiments, HaCaT cells were seeded
to stop the detaching process and cells were suspended with in a 6-well cluster plate (150,000300,000 cells/2 mL well)
stronger shaking, followed by the use of a syringe (21 G) and incubated 24 h under the same culture conditions, as
because of its mechanical resistance. All cell manipulation mentioned above. After this period, the cells were exposed to
and growth and subsequent exposure conditions were per- complexed lycopene (10 M) for 24 h in order to achieve its
formed under strict aseptic procedures. cellular internalization and a higher cell confluence, and after
Cell density was calculated by counting with a hemo- UV-B irradiation (225 mJ/cm2 ) cells were allowed to grow
cytometer (Neubauer Improved) under a phase-contrast under standard culture conditions for another 24 h to enable
microscope Nikon Eclipse 80i (Coyoacan, Mexico). the occurrence of cellular repair mechanisms.
Cell Adhesion Internalization UV-B Recovery Assay

DMEM, 24 h Exposure -MEM, 24 h DMEM, 24 h
seeding irradiation detection
10 M Lyc-CD 225 mJ/cm2 (i) Cytotoxicity

(ii) Genotoxicity
(iii) Apoptosis
(iv) ROS levels
Figure 1: Culture conditions and experimental setting for the study of HaCaT cells exposed to complexed lycopene (Lyc-CD) and UV-B
irradiation.
2.5. MTT Assay. Cell metabolic activity was assessed by the to G0/G1 was positioned at channel 200. This setting was
MTT (Sigma-Aldrich, St. Louis, MO, USA) assay, which is kept constant. The results were obtained in the form of three
often used to roughly estimate cultures viability/proliferation graphics: linear fluorescence light intensity (FL), forward
characteristics. Cells were seeded at a density of 7,000 angle light scatter (FS) versus side angle light scatter (SS), and
cells/well in a 96-well cluster plate. After complexed lycopene FL pulse integral versus FL pulse height. This last cytogram
exposure, UV-B irradiation, and 24/h recovery the MTT was used to eliminate partial nuclei and other debris, nuclei
assay was performed as previously described [46]. The optical with associated cytoplasm and doublets (these events have a
density of reduced MTT was measured at 570 nm by spec- higher pulse area but the same pulse height as single nuclei).
troscopy on an automatic microtiter plate reader (Synergy Cell cycle analysis was performed using the FlowJo software
HT Multi-Mode from BioTeK Instruments Inc., Winooski, (Tree Star Inc., Ashland, Oregon, USA) applying the Dean-
VT, USA) and cell metabolic activity (MA viability or Jett Model. Results were expressed as % of nuclei in G0/G1, S,
proliferation characteristics) was calculated according to and G2 phases of the cell cycle. The % of nuclei in Sub-G0/G1
indicative of DNA fragmentation was also computed.
Cell MA (%)
2.7. Analysis of Apoptosis by Flow Cytometry. Early and late
(Abs 570 nmsample Abs 570 nmDMSO ) apoptosis were investigated by FCM using Annexin V-FITC
= (1)
(Abs 570 nmnegative control Abs 570 nmDMSO ) Apoptosis Detection Kit from BD Pharmingen (Franklin
Lakes, NJ, USA), as previously described [47]. Briefly,
100. after complexed lycopene exposure and UV-B irradiation
as described before, the cells were gently harvested with
Besides the negative control (nonexposed and nonirradiated Accutase (PAA Laboratories, Pasching, Austria) and washed
cells), the assays results were also compared to CD aqueous twice in cold PBS (1 mL). Finally, Annexin V-FITC and PI,
solutions (vehicle control) with the same dilution factors used 5 L of each, were added to 100 L of cell suspension (105
for complexed lycopene samples. cells/mL) in binding buffer. Samples were left in the dark for
15 min and 400 L of binding buffer was added. Samples were
2.6. Cell Cycle Analysis by Flow Cytometry. After complexed analyzed by FCM using a Coulter EPICS XL flow cytometer
lycopene exposure and UV-B irradiation as described before, (Coulter Electronics, Hialeah, Florida, USA). Data were
the cells were harvested with Accutase and centrifuged acquired using the SYSTEM II (v. 2.5) software. The cytogram
at 1157 g for 5 min at 4 C. The supernatant was removed of FITC fluorescence in log scale versus PI fluorescence in log
and the cells were washed in PBS and suspended in 1 mL scale allows the identification of nonapoptotic cells (Annexin
of 85% ethanol at 4 C and kept at 20 C until analysis. V-FITC negative, PI negative), exclusively early apoptotic
After that, the cells were centrifuged twice at 1157 g for cells (Annexin V-FITC positive, PI negative), predominantly
5 min at 4 C and suspended in 800 L PBS and vortexed. late apoptotic cells (Annexin V-FITC positive, PI positive),
Cell cycle analysis was done as previously described [46]. and predominantly necrotic or dead cells (Annexin V-FITC
Briefly, samples were filtered on a nylon mesh (50 m pore negative, PI positive). FCM data were analyzed by FlowJo
size) to the analysis tubes. Propidium iodide (PI), a DNA software (Tree Star Inc., Ashland, OR).
intercalating fluorochrome, and RNase were added to the
samples (50 L each one) and vortexed. The mixture was 2.8. RNA Extraction and qPCR Analysis of
incubated 20 min at room temperature and then analyzed Apoptosis Regulating Genes
by flow cytometry (FCM) using a Coulter EPICS XL flow
cytometer. The instrument was equipped with an air-cooled 2.8.1. Total RNA Extraction. At least 1 105 HaCaT cells
argon-ion laser tuned at 15 mW and operating at 488 nm for were rinsed twice with sterile PBS after removal of culture
excitation. Data was acquired using the SYSTEM II (v. 2.5) medium. After rinsing, cells were lysed in 1 mL TRIzol
software. Integral fluorescence together with fluorescence reagent (Life Technologies, Saint Louis, MO, USA) and
pulse height and width emitted from nuclei was collected after 5 min room temperature incubation, 200 L chloroform
through a 645 dichroic long-pass filter and a 620 band-pass was added to each sample, shaken on vortex for 10 s, and
filter and converted on 1024 ADC channels. Prior to analysis incubated at room temperature for 2 min. Phase separation
the amplification was adjusted so that the peak corresponding was achieved by centrifugation at 12,000 g for 5 min at 4 C
in Phase-Lock Gel Heavy tubes (5 Prime 3 Prime, Inc., cytometer and ROS formation was estimated by the median
Boulder, CO, USA). The aqueous phase was mixed with 1 fluorescence intensity (MFI) parameter using the FlowJo
volume 70% ethanol and RNA was further purified using software.
RNeasy Mini Kit columns (Qiagen, Hilden, Germany) fol-
lowing the manufacturers recommendations. The total RNA 2.10. Statistical Analysis. The results are reported as mean
was quantified by spectrophotometry at 260280 and 230 SD of at least three replicates/treatment. In addition, at
260 nm (Nanodrop Spectrophotometer ND-1000, Thermo least three independent assays were performed for each
Fisher Scientific, Wilmington, DE, USA). analysis. For gene expression analysis, results are reported as
mean SE of at least three replicates from 2 independent
2.8.2. cDNA Synthesis. 1 g total RNA was reverse-tran- assays. The results of all these experiments were statistically
scribed using the Omniscript RT Kit (Qiagen, Hilden, Ger- analyzed by Analysis of Variance (ANOVA with All Pair-
many) in a reaction mixture containing 1 M Oligo(dT)18 wise/Nonpairwise Multiple Comparison Procedures) using
primer, 5 mM dNTPs, reaction buffer, and RT enzyme SigmaPlot 11.0 software. The differences were considered
according to the manufacturers instructions. After the enzy- statistically significant when < 0.05.
matic reaction (incubation at 37 C for 1 h), cDNA samples
were prediluted in milliQ water (1 : 20). 3. Results
2.8.3. Quantitative RT-PCR (qPCR). For qPCR, primers were 3.1. HaCaT Cell Growth and Confluence under Normal Cul-
used complementary to the genes coding for Bax, Bcl-2, ture Conditions. HaCaT cell growth and confluence under
caspase 3, and TRAIL proteins, respectively, BAX, BCL2, normal culture conditions until 120 h are represented on
CASP3, and TNFSF10. SDHA (succinate dehydrogenase) Figure 2(a). As it can be observed, the exponential phase
was used as the reference gene. The target genes and extends until approximately 72 h and the full confluence can
corresponding oligonucleotide primer sequences (5 to 3 ) be maintained more than 1 week. The selected confluence
were as follows: BAX-F: GACGGCCTCCTCTCCTACTT; for complexed lycopene exposure and UV irradiation in this
BAX-R: CAGCCCATCTTCTTCCAGAT; BCL2-F: GGA experiment was attained at 24 h and 48 h, respectively.
GGATTGTGGCCTTCTTT; BCL2-R: GCCGGTTCAGG Under phase-contrast microscopy, the cells displayed typ-
TACTCAGTC; CASP3-F: GAACTGGACTGTGGCATT ical intermediate phenotype of polygonal cells interspersed
GA; CASP3-R: TGTCGGCATACTGTTTCAGC; TNFSF10- with giant often multinucleated cell and single morphology
F (TRAIL-F): CCTGCAGTCTCTCTGTGTGG; TNFSF10- (Figure 2(b)).
R (TRAIL-R): ACGGAGTTGCCACTTGACTT; SDHA-
F: CTGCAGAACCTGATGCTGTGT; SDHA-R: GGATGG- 3.2. Effect of UV-B Doses on Cell Metabolic Activity by
GCTTGGAGTAATCG. MTT Assay. As theoretically expected, increasing UV-B dose
Primer design was performed using Primer3 [48] and resulted in decreased cell metabolic activity (Figure 3). UV-
primer specificity was confirmed using the In-Silico PCR B irradiation resulted in distinct morphological changes in
UCSC Genome Browser [49]. The final individual qPCR HaCaT cells, as irradiated cells became round and detached
reactions contained iTaq Universal SYBR Green Supermix from the surface of the plate.
(BioRad, Hercules, CA), 150 nM of each primer, and 1 : 4 (v/v) According to MTT results, the UV-B condition tested
prediluted cDNA (1 : 20). Two qPCR technical replicates were that was near but above 50% viability was 225 mJ/cm2 . This
performed per sample in the iQ5 Bio-Rad thermal cycler. The UV-B dose was chosen as high UV-exposure condition to
qPCR program included 1 min denaturation at 95 C, followed analyze the maximum range of effects and response to UV
by 60 cycles at 94 C for 5 s, 58 C for 15 s, and 72 C for 15 s. exposure.
After qPCR, a melting temperature program was performed. Complexed lycopene (Lyc-CD) up to 15 M did not affect
Mean PCR efficiencies and cycle thresholds were determined the metabolic activity of nonirradiated cells, and only 20 M
from the fluorescence data using the algorithm Real-Time Lyc-CD led to significant decrease in metabolic activity in
PCR Miner [50]. Relative gene expression of cell samples these cells (Figure 4). At doses equal to or higher than 15 M,
relative to SDHA was calculated using the Pfaffl method [51]. complexed lycopene decreases the MA of irradiated cells
(225 mJ/cm2 ), compared to cells not preexposed to lycopene
2.9. Reactive Oxygen Species (ROS) Quantification by (Figure 4).
Flow Cytometry. ROS (O2 and OH) generation was According to preliminary MTT assays, it was also
assayed by FCM using the fluorescent probe 2,7-dichlo- observed that concentrations of Lyc-CD higher than 20 M
rodihydrofluorescein diacetate (H2 DCF-DA), as described induced a higher decrease in MA in irradiated cells (supple-
previously [47], which upon acetate cleavage is oxidized to mentary data in Supplementary Material available online at
fluorescent dichlorofluorescein (DCF) by hydrogen peroxide. http://dx.doi.org/10.1155/2016/8214631).
After complexed lycopene exposure and UV-B irradiation as According to these results, we decided to choose an inter-
described before, the medium was replaced by serum-free mediate complexed lycopene concentration (10 M) whose
-MEM containing 10 M H2 DCF-DA for 30 min, at 37 C effects have been previously established in other cell lines [52
in dark. Cells were washed with PBS, trypsinized with 54]. At this lycopene concentration, the cell metabolic activity
Accutase collected, and analyzed in a Coulter EPICS XL flow was higher than 50% and was not significantly different from
120
Cell confluence (%) 100
80
60
40
20
0
0 20 40 60 80 100 120 140
Time (hours)
(a) (b)
Figure 2: (a) HaCaT cells growth and confluence curves under normal culture conditions for 120 h. (b) HaCaT cells observed by phase-
contrast microscope (100x magnification), scale bar: 20 m.
MTT assay MTT assay

100 120
A A
A A A A A
100 A
80
B
B, C C B B B
80
60
MA (%)
MA (%)
D 60
40
40
20
20
0 0
75 150 200 225 325 0 5 10 15 20
UV-B irradiation dose (mJ/cm2 ) Lycopene concentration (M)
Figure 3: Effect of different UV-B doses on cell metabolic activity NI

(MA) determined by MTT assay. Results are expressed as percentage IR
(mean SD) of cell MA compared to nonirradiated cells. Statistical
analysis: One-Way ANOVA with All Pairwise Multiple Comparisons Figure 4: Effect of complexed lycopene preexposure on UV-B-
by Holm-Sidak method: statistical differences between the samples irradiated (IR, 225 mJ/cm2 ) and nonirradiated (NI) HaCaT cells on
are represented by different letters when < 0.05. cell MA measured by MTT assay. Results are expressed as percentage
(mean SD of 3 independent experiments with 6 replicates each
one). Statistical analysis: One-Way ANOVA with Multiple Com-
parisons versus Control Group (Holm-Sidak method): statistical
the nonexposed cells. Higher lycopene concentrations could differences between the samples within nonirradiated and irradiated
decrease the cell viability, for example, due to a prooxidant groups (in respect to cells not exposed to lycopene) are represented
effect, as suggested by a decrease in viability compared to by different letters when < 0.05.
nonexposed, nonirradiated cells.
3.3. Cell Cycle Analysis. Figure 5 shows representative his- 0.008, resp.) (Figure 5). Although the S and G2 phases were
tograms of cell cycle of HaCaT cells after 10 M complexed not significantly affected by any of the treatments, an increase
lycopene exposure and UV-B irradiation (225 mJ/cm2 ). Cell in S-phase frequency can be observed.
cycle analysis shows that complexed lycopene exposure alone As previously observed in Figure 5, the analysis of PI-
did not significantly ( > 0.05) affect the dynamic of cell cycle stained nuclei by FCM cell cycle analysis showed an increase
in comparison to control cells. Compared to nonirradiated in sub-G1 subpopulations upon UVB irradiation. Exposure to
and nonexposed cells, irradiation induced a decrease in the CD vehicle induced a small decrease in % sub-G1 irradiated
percentage of cells in the G0/G1 phase of cell cycle especially cells, compared to nonexposed, irradiated cells. Contrarily to
in complexed lycopene and CD exposed cells ( = 0.011 and cells exposed to CD, cell exposed to Lyc-CD did not present
G0/G1
NI NI, CD NI, Lyc-CD
400 400 400
300 300 300

Count
Count
Count
200 200 200
G2
100 100 100
S
0 0 0
0 200 400 600 0 200 400 600 0 200 400 600
FL3 FL3 FL3
IR IR, CD IR, Lyc-CD

400 400 400
300 300 300

Count
Count
Count
200 200 200
Sub
100 G1 100 100
0 0 0
0 200 400 600 0 200 400 600 0 200 400 600
FL3 FL3 FL3
(a)
90
A, B
80
A A, B
70 A, B
60
C B, C
Cells (%)
50
40
B#
30 B#
20 A, B#
10 A# A# A#
0
NI
NI, CD
NI, Lyc-CD
IR, CD
IR, Lyc-CD
IR
Sub-G0/G1 S
G0/G1 G2
(b)
Figure 5: Effect of lycopene and UV-B on the cell cycle: (a) cell cycle histograms of UV-B (225 mJ/cm2 ) irradiated (IR) and nonirradiated
(NI) HaCaT cells exposed to 10 M complexed lycopene (Lyc-CD) and to the respective controls; (b) cell cycle phase distribution of UV-B
(225 mJ/cm2 ) irradiated (IR) and nonirradiated (NI) HaCaT cells exposed to 10 M complexed lycopene (Lyc-CD) and the respective controls,
including cyclodextrin vehicle (NI, CD and IR, CD). Results are expressed as percentage (mean SD). Statistical analysis: One-Way ANOVA
with All Pairwise Multiple Comparison Procedures: means with different letters (A, B, and C) are significantly different ( < 0.05). In this
case, only the statistical differences between groups presenting differences (G0/G1 and sub-G1) were marked for simplification purposes.
Comparison between % cells with sub-G1 amount of DNA (#) and % cells in G0/G1 phase () is presented.
small decrease in % sub-G1, compared to nonexposed, irra- production, two cell populations were observed in irradiated
diated cells. cells against one in nonirradiated cells. The Median Fluo-
rescence Intensity (MFI) from irradiated samples was statis-
3.4. Apoptotic Markers. Annexin V assay differentiates tically higher than MFI from correspondent nonirradiated
subpopulations that are necrotic, apoptotic, or viable. cells, as theoretically expected. Complexed lycopene did not
Comparative analysis of these subpopulations in irradi- increase ROS production in nonirradiated cells. However, in
ated (225 mJ/cm2 ) and nonirradiated HaCaT cells, treated irradiated cells Lyc-CD induced a significant ROS increase
with 10 M complexed lycopene, was performed using the compared to irradiated nonexposed cells.
Annexin V assay (Figure 6).
Within this study, a high UVB dose was used to induce 4. Discussion
cell alterations and apoptosis, but not excessive necrosis. For In this work, we used HaCaT cells, a spontaneously trans-
this study only the adherent cells were analyzed, which means formed human epithelial cell line from adult skin which
that the necrotic cells in suspension were not considered maintains full epidermal differentiation capacity. This cell line
in the assay results. Nevertheless, as shown in Figure 6, the is immortal (>140 passages) but remains nontumorigenic,
main shift upon UVB irradiation was from viable to apoptotic and it is aneuploid (hypotetraploid) with unique stable
cells, confirming that the high UVB dose used was not marker chromosomes indicating monoclonal origin [40]. As
excessively detrimental for the HaCaT cells attached to plate performed in this experimental work, further investigation
and used in all the assays described here. In nonirradiated
needs to include studies dealing with normal cells, their
cells, complexed lycopene did not affect ( > 0.05) the
transformation into malignant cells, and the association
percentage of necrotic, apoptotic, and viable cells compared
to the control (nonexposed cells). Contrarily, UV-B irradi- between malignant cells and the surrounding normal cells in
ation alone increased the percentage of both early and late order to determine the cytotoxicity in both cell populations.
apoptotic cells ( < 0.05) and decreased the percentage of It is noteworthy that cell culture studies are usually
viable cells compared to the control. Contrarily to what was carried out under abnormal conditions known as culture
observed for nonirradiated cells, the Annexin V assay showed shock, where cells are exposed to high oxygen tension and to
a decrease in the % cells with Annexin V positive staining free metal ions in the medium [27, 55]. Thus, it must always be
(early and late apoptosis) preexposed to complexed lycopene taken into account if the study compound reacts with the cell
in irradiated cells ( < 0.05), while the % necrotic cells medium. Different half-life values of lycopene under standard
increased. cell culture are reported in the literature, for example, 12
Also complementary data showed that irradiated cells 20 h [37]. Experiments were conducted with exposure time
previously exposed to the vehicle (CD) presented, once of 24 h which was sufficient to reveal the cellular effects of
again, results quite similar to those of nonirradiated cells Lyc-CD. Lycopene chemical stability was conferred by CD
(supplementary data). complexation.
The antioxidant action of carotenoids is related to their
3.5. Effect of Complexed Lycopene on the Expression of Anti- ability to trap free radicals and quench singlet oxygen.
and Proapoptotic Genes. Under UVB irradiation, lycopene However, depending on the redox potential of lycopene and
complex exerted proapoptotic effects compared to irradiated the surrounding environment, its antioxidant activity may
but not exposed cells (Figure 7). Compared to irradiated shift to prooxidant activity [56, 57]. In fact, the HaCaT cells
and nonexposed cells, in irradiated cells the exposure to medium (DMEM) contains ferric nitrate (Fe(NO3 )3 9 H2 O)
vehicle CD inhibited antiapoptotic BCL2 expression but and Fe(III) can react with excess neutral carotenoids, such
did not increase proapoptotic BAX expression. Contrarily as lycopene. Ferric ions have been proposed to degrade
to this, in irradiated cells Lyc-CD increased proapoptotic carotenoids by the following mechanism [58, 59]: Fe3+ +
BAX expression but did not inhibit antiapoptotic BCL2
carotenoid Fe2+ + carotenoid+ . Although there are many
expression. This observation points to a proapoptotic effect
of Lyc-CD mediated by BAX upregulation. Moreover, in Fe chelators that inhibit the reactions of Fe, oxygen, and their
irradiated cells Lyc-CD showed lower caspase 3 gene (CASP3) metabolites [60], these chelating agents may also mediate tox-
gene expression compared to nonexposed, irradiated cells; icity by stimulating Fe-mediated oxygen radical generation
however, it increased CASP3 gene expression comparatively [61]. The chelating agent used for HaCaT trypsinization was
to CD vehicle. TRAIL is a proapoptotic cytokine secreted by ethylenediaminetetraacetic acid (EDTA) which can induce
many cell types; however, in this study, UVB light was found Fenton-chemistry mediated radical damage. In fact, the
to decrease TRAIL expression. autoxidation of Fe(II) enhanced by EDTA was observed
by others [57, 58]. Therefore, in order to prevent lycopene
3.6. Reactive Oxygen Species (ROS) Analysis. Analysis of oxidation by Fe (III), another cell culture medium (-MEM)
oxidative stress induction in HaCaT cells after 10 M com- was used without this element during cells exposure to
plexed lycopene exposure and UV-B irradiation (225 mJ/ lycopene. A normal cellular growth was observed. Further-
cm2 ) was performed by determining reactive oxygen species more, it should be also noted that the solution with the
(ROS) formation by FCM. highest lycopene dose had a suitable osmolarity lower than
The results of irradiated and nonirradiated cells are 320 mOsm/kg (268 mOsm/kg).
represented in Figure 8. UVB irradiation induced an increase During storage, special precautions were taken such as
in ROS intracellular production. In fact, regarding ROS the protection of lycopene from temperature, light, and
NI NI, Lyc-CD
103 103
FL3 LOG: PI LOG
FL3 LOG: PI LOG

102 102
101 101 100

90 A
100 100 AC
80 AC
1 1
10 10
70
101 100 101 102 103 101 100 101 102 103 B
FL1 LOG: FITC LOG FL1 LOG: FITC LOG 60
Cells (%)
50
IR IR, Lyc-CD
103 103
40
FL3 LOG: PI LOG
FL3 LOG: PI LOG
2 2 B
10 10 30
B
20
101 101 A A AB AB
10 A A
ABAB B
100 100 A
0
Necrotic Late apoptotic Early apoptotic Viable and
1 1
10 10 nonapoptotic
101 100 101 102 103 101 100 101 102 103 NI IR
FL1 LOG: FITC LOG FL1 LOG: FITC LOG NI, Lyc-CD IR, Lyc-CD
(a) (b)
Figure 6: (a) Representative examples of Annexin V-FITC Dot-Plots Gating (FL1 LOG versus FITC LOG) of nonirradiated (NI) and UV-B
(225 mJ/cm2 ) irradiated (IR) HaCaT cells not previously exposed or exposed to 10 M complexed lycopene (Lyc-CD); (b) results are expressed
as percentage (mean SD, = 3) of nonapoptotic and viable cells, Q8: Annexin-FTIC () and PI (); early apoptotic cells, Q5: Annexin-FTIC
(+) and PI (); predominantly late apoptotic or dead cells, Q6: Annexin- FTIC (+) and PI (+); and predominantly necrotic and dead cells,
Q7: Annexin-FTIC () and PI (+). Statistical analysis: One-Way ANOVA with All Pairwise Multiple Comparison Procedures (Holm-Sidak
method): means with different letters are significantly different ( < 0.05).
air. However, during lycopene exposure, cell culture was UV-B irradiation activates diverse cellular responses in
maintained at 37 C in a humidified incubator with 5% CO2 human cells, such as cell cycle arrest, DNA repair, and apop-
atmosphere. Even considering other experimental conditions tosis through signal transduction pathways [15]. Previous
reported in the literature [62] that comprise the addition studies have shown that UVR can cause cell cycle arrest both
of lycopene solutions to the cell culture medium under N2 at G1 [63] and G2 phases [64] of cell cycle. However, in our
environment, we preferred to maintain our protocol to avoid study, irradiation did not affect significantly the percentage
perturbing the cell culture with other variables. of cells on S and G2 phases (Figure 5). On the contrary, the
The level of lycopene normally observed in human percentage of cells in G1 phase decreased in irradiated cells,
plasma is on the order of 0.5 M even with dietary supple- especially those previously exposed to complexed lycopene
mentation [20, 52, 53]. Therefore, for therapeutic purposes or the respective vehicle alone (cyclodextrins). Cyclin D
(assuming topical application) we used a range above these regulates the transition from G0 to early G1 phase, while
normal plasma levels (5 up to 20 M). According to MTT cyclin E regulates the transition of the cell from late G1 phase
results, 20 M is revealed to be toxic in in vitro studies to S phase; p21 and p27 CDK inhibitors bind and inhibit
(Figure 4). Thus, 10 M lycopene nominal concentration was the activity of cyclin E/CDK2 complex, blocking cell cycle
selected for further studies, supporting the selected dose used progression in G1 phase. In fact, after UV irradiation, the
in other in vitro studies [5254]. half-life of the tumor suppressor (p53) appears to be extended
The option to use cyclodextrins for lycopene solubiliza- which will induce the p21 CDK inhibitor leading to G1 phase
tion and photoprotection was based on data resulting from arrest or cell death by apoptosis. G2-phase checkpoint control
previous studies (Table 1) on parameters/conditions affecting does not appear to be affected [65]. It has been reported
the delivery of lycopene to cells, including the solubilization, that the growth inhibition of lycopene on MCF-7 breast
stabilization, and cellular uptake by using other vehicles. In cancer cells was also associated with decreased G1-S cell cycle
addition, similar MTT results were obtained with another progression, decreased cyclin D1 expression, and stabilization
vehicle (supplementary data). of p27 in the cyclin E-CDK complex [7, 66]. Cell cycle
HaCaT keratinocytes have a mean intermitotic time of arrest increases the time available for DNA repair before its
2224 h in vitro [13] and their cell cycle is subjected to replication and mutation fixation processes [5]. Regarding the
regulation by cyclins, cyclin-dependent kinases (CDKs), and cell cycle results obtained with CD, it has been reported that
CDK inhibitors. methyl -CD inhibits cell growth and induces cell cycle arrest
BAX BCL2
6 12E 3

5 10E 3
4 8E 3
Normalized expression
3 6E 3
2 4E 3
1 2E 3
0 0

Control +CD +Lyc-CD Control +CD +Lyc-CD
CASP3 TRAIL
14E 1 1.4

12E 1 1.2

10E 1 1.0


8E 1 0.8
6E 1 0.6
4E 1 0.4
2E 1 0.2
0 0

Control +CD +Lyc-CD Control +CD +Lyc-CD
NI NI
IR IR
Figure 7: Representation of Bax, Bcl-2, caspase 3, and TRAIL gene expression values in HaCaT cells exposed to 10 M complexed lycopene
(Lyc-CD) normalized to the SDHA reference gene. Cells were irradiated with 225 mJ/cm2 UV-B (IR) and nonirradiated (NI). Results are
expressed as mean SEM of three technical replicates from two independent assays.
via a prostaglandin E2 independent pathway in Raw264.7 p53 alleles) are more susceptible to apoptosis than normal
macrophage cells [67]. Thus, a control assay with CD alone keratinocytes in vitro, possibly because of aberrant signaling
is mandatory in these studies. pathways resulting from long-term culture [69, 70].
Apoptosis is the best-characterized type of programmed In this work, a decrease in % apoptotic cells was observed
cell death because of its importance in development, home- in irradiated cells preexposed to Lyc-CD (Figure 6). However,
ostasis, and pathogenesis of different diseases, such as cancer. this result must be interpreted cautiously, as CD has been
Cells respond to specific apoptotic signals by initiating intra- previously reported to deplete cholesterol from cell mem-
cellular processes that result in typical physiological changes. branes [71, 72]. Cholesterol presence is essential for lipid
Among these changes are externalization of phosphatidylser- raft formation and Fas-receptor activation, and this could
ine to the cell surface, depolarization of mitochondrial mem- inhibit UV-B induced apoptosis, as demonstrated by George
branes, cleavage and degradation of specific cellular proteins, et al. [73]. Depletion of cholesterol by methyl--cyclodextrin
compaction and fragmentation of nuclear chromatin, loss of reduced Fas aggregation which was accompanied with a
cell membrane integrity, and cellular shrinkage. We studied reduced apoptotic but increased nonapoptotic death of UV-
one of the earliest apoptotic events, that is, the translocation B-irradiated HaCaT cells [73]. In preliminary experiments,
of phosphatidylserine (PS) from the inner to the outer leaflet we in fact observed that CD alone can decrease the %
of the plasma membrane. Moreover, Bowen et al. [68] have apoptotic cells (supplementary data). Moreover, since CD can
found that HaCaT cell line (which harbors two mutant form complexes with lipids from the plasma membrane, it
80
C, B
60 B
MFI
40
A
A
1 0 1 2 3 20 A
10 10 10 10 10
FL1 LOG:: FL1 LOG
IRR, Lyc-CD
IRR, CD 0
NI
NI, CD
NI, Lyc-CD
IRR, CD
IRR, Lyc-CD
IRR
IRR
NI, Lyc-CD
NI, CD
NI
(a) (b)
Figure 8: Intracellular ROS quantification: (a) FL1 histogram plots, showing the distribution of cell count versus DCF fluorescence, and
(b) Mean Fluorescence Intensity (MFI) histograms results of HaCaT UV-B irradiated cells, IR (225 mJ/cm2 ), and nonirradiated cells (NI)
exposed to 10 M complexed lycopene (Lyc-CD) and to the respective controls labelled with DCF-DA. Statistical analysis: One-Way ANOVA
with Multiple Pairwise Comparisons: medians with different letters are significantly different ( < 0.05).
might be possible that PS was shielded by CD against binding A more close focus on BAX and CASP3 expression reveals
to Annexin V, a requirement for the validity of Annexin that lycopene induced increased BAX expression in cells
V assay. Nevertheless, cells exposed to Lyc-CD showed an treated with the high UVB dose used in this study and this
increase in the Bax/Bcl2 gene expression ratio. Moreover, might represent an important mechanism for its therapeutic
Lyc-CD completely reverted the inhibitory effect of CD in action.
TRAIL expression. These results suggest a proapoptotic effect Besides apoptosis, necroptosis, and necrosis or senes-
of lycopene which is not found in CD. UV-B irradiation cence, damaged keratinocytes can also be physiologically
has been shown to increase levels of apoptosis biomarkers, eliminated by induction of terminal differentiation. This
especially the proapoptotic protein Bax, thereby inducing may be readily monitored by detection of, for example,
apoptosis in skin cells. Regarding the apoptosis biomarkers, suprabasal keratins or cytoplasmic involucrin for late steps
Bcl-2 family proteins can modulate mitochondrial perme- in epidermal differentiation; however, this type of analysis
ability through oxidative phosphorylation during apoptosis. would probably be most useful for very late time points. It
Changes in the Bax/Bcl-2 ratio suggest a corresponding should be noted that more biomarkers could be also analyzed
change in mitochondrial permeability to release apoptogenic considering the huge cascade events triggered by UVR, for
molecules from the mitochondria to the cytosol [5]. In this example, protein kinase C family which sensitizes skin to
work, irradiated cells showed significantly higher levels of UVR [74] and others regarding the influence on intracellular
apoptosis, confirmed by independent parameters (namely, calcium levels or retinol signaling which are profoundly
increased sub-G1 population indicative of DNA fragmen- altered upon differentiation of keratinocytes [75].
tation) and binding to Annexin V, indicative of increased UVR results in an increased generation of ROS that
phosphatidylserine present in the outer leaflet of the cell interacts with proteins, lipids, and DNA, overwhelming the
membrane. From the gene expression data obtained, this antioxidant defense mechanisms of the cells.
means that the most relevant parameters to take into account The epidermis is composed mainly of keratinocytes,
for UVB-induced apoptosis are BAX and CASP3 expression. which are rich in ROS detoxifying enzymes such as super-
BCL2, a known antiapoptotic effector was found upregulated oxide dismutase, catalase, and glutathione peroxidase and
in this study, and TRAIL, a proapoptotic cytokine, was found in low-molecular-mass antioxidant molecules [76]. Although
downregulated under UVB irradiation. This suggests that skin spontaneously responds to increased ROS levels, this
under the experimental conditions used, these genes have a response may not be sufficient to prevent the progression of
comparatively lower predictive value for apoptosis induction skin cancer [4]. Despite the extensive evidence implicating
by UVB. It might be a response at transcription level which ROS in oxidative DNA damage, little is known about its
does not reflect the physiological level of the irradiated cells. involvement in DNA damage of keratinocytes, which are the
most relevant cell type in nonmelanoma skin cancer. The 5. Conclusions

singlet oxygen and hydroxyl radicals are the major damaging
oxidative species which can be formed under normal aerobic According to data obtained from all experimental assays, we
metabolism and by certain processes including photosensi- demonstrate here that complexed lycopene up to 10 M does
tization [77]. The major DNA oxidation products include 8- not show metabolic toxicity (MTT assay) under standard cell
oxo-7-hydro-deoxy-guanosine (8-oxodG) and 2,6-diamino- culture conditions. On one hand, at nontoxic dose (10 M)
4-hydroxy-5-formamidopyrimidine [78]. complexed lycopene does not affect the profile of apoptotic,
Comparatively to nonirradiation, UV-B irradiation sig- necrotic, and viable cells or show cytostatic effects despite
nificantly altered the distribution of cells in terms of fluo- slightly increasing the ROS content. However, cells previ-
rescence (Figure 8). In irradiated cells two populations can ously exposed to complexed lycopene when irradiated with
be distinguished. The first peak might correspond to dead metabolically damaging UV-B dose show a distinguishing
cells, which had a very weak signal of DCF (compatible switch in the dead : apoptotic : viable subpopulations com-
with autofluorescence). In this case, the DCF might not be pared to nonexposed irradiated cells. On the other hand,
activated and metabolized within the cell (no fluorescence), exposed irradiated cells showed a decrease in G0/G1 phase.
and/or the cells might not retain the DCF because of the In fact, the increased sub-G0/G1 phase and a trend for S-
loss of membrane integrity. Complexed lycopene increased phase delay (even not statistically different) could contribute
the ROS levels in irradiated cells. Reagan-Shaw et al. [5] to this cell cycle change. In addition, an increased expression
have also obtained a significant decrease of SOD activity of different apoptosis biomarkers (BAX and caspase 3) was
(MnSOD) after treatment with sanguinarine, enhancing observed in exposed irradiated cells when compared to the
UV-B-mediated oxidative stress in HaCaT cells. However, respective controls. These two biomarkers were the most
according to Onoue et al. [79], ROS data might not always useful probably because those genes might be more involved
provide a reliable indication for the capacity of a chemical in this process.
to participant in a photogenotoxic cascade. This fact could Therefore, complexed lycopene might play a corrective
be more realistic when irradiation is used in experiments. role or cytotoxic effect in photodamaged and preneoplastic
In fact, the photodegradation of lycopene may contribute keratinocytes, while allowing other keratinocytes to acceler-
to cytotoxicity, including oxidative damage. For example, ate repairing mechanisms becoming viable. However, some
apo-6 -lycopene and 2-methyl-hepte-6-one as well as further results could be altered by the CD interference with some
reaction products are formed during irradiation of lycopene in vitro assays and also with the cells particularly after a
[28, 80]. Nagao [81] found that oxidized metabolites of high damage effect. Although CDs seemed to be a good
lycopene but not lycopene itself can inhibit cell growth candidate to vehicle lycopene in order to counterbalance
and stimulate apoptosis in a different cell line (HL-60). the disadvantages of most solvents usually used, here we
In another study, lycopene in human prostate cancer cells found that it was not suitable under this protocol conditions,
inhibited cell growth, but the oxidized mixtures displayed especially UV irradiation. Nevertheless, these are interesting
markedly more potent growth inhibition [81, 82]. On the data regarding the CD effect on cell cultures and reveal the
contrary, some recent studies suggested that lycopene or importance of the analysis of this control compared to cells
lycopene metabolites may, as -carotene and its metabolites exposed to active molecules, as it was here performed.
do, enhance carcinogenesis. Future studies will continue with other exposure condi-
In general, similar results were obtained by Reagan-Shaw tions, that is, another lycopene vehicle and a lower UV-B dose
et al. [5] who suggested that sanguinarine (also a botanical which could be set by other assays, such as TUNEL and/or
antioxidant) may protect skin cells (also HaCaT cell line) detection of chromosomal breakage by Cytokinesis-Blocked
from UVB-mediated damage via apoptotic elimination of Micronucleus Cytome Assay (CBMN) (besides MTT). More-
damaged cells that escaped from the programmed cell death. over, detection of Ki67 protein, a marker of proliferation,
These are clearly important observations since apoptosis is could be attempted. The analysis of other biomarkers and
a mechanism of defense and acts by opposing the creation final studies using 3D skin models as previously suggested
of damaged and preneoplastic cells and expansion to a would resemble more closely the situation in vivo, which may
clone. Once mutations arise, apoptosis also removes preneo- give definite and clear answers.
plastic cells that are aberrantly proliferating due to genetic
defects. Conflict of Interests
However, further investigation into the dose effect of
lycopene and further understanding of the metabolism of All authors have no conflict of interests to declare.
apo-10 -lycopenoids on carcinogenesis are still needed [7].
On the other hand, studies on skin organotypic 3D-cocultures Acknowledgments
(long-lived 3D-cocultures, epiSC-markers, label-retaining
cells, etc.) would be useful to have all picture considering This work has been funded by the European Regional
the loss of complex interactions between the epithelium and Development Fund (FEDER) through the Competitive Fac-
stroma in 2D cell cultures [83]. An UV-irradiation protocol tors Thematic Operational Programme (COMPETE) and
for a normal skin model or even the malignant model for both by National Funds through the Foundation for Science
prevention and treatment approaches of skin cancer could be and Technology (FCT), under the projects PEst-OE/SAU/
used [84, 85]. UI4013/2011 and UID/AMB/50017/2013. The grants awarded
by FCT to Helena Oliveira (SFRH/BPD/48853/2008) and Jose caffeine, Journal of Investigative Dermatology, vol. 129, no. 7, pp.
Miguel P. Ferreira de Oliveira (SFRH/BPD/74868/2010) are 18051815, 2009.
also acknowledged. The authors acknowledge Dr. Armando [16] Z. Fazekas, D. Gao, R. N. Saladi, Y. Lu, M. Lebwohl, and H. Wei,
Costa for technical support. Protective effects of lycopene against ultraviolet B-induced
photodamage, Nutrition and Cancer, vol. 47, no. 2, pp. 181187,
2003.
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http://dx.doi.org/10.1155/2016/6859523
Review Article
Mitochondrion-Permeable Antioxidants to
Treat ROS-Burst-Mediated Acute Diseases
Zhong-Wei Zhang,1 Xiao-Chao Xu,2 Ting Liu,3 and Shu Yuan1

1
College of Resources Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
2
College of Bioindustry, Chengdu University, Chengdu 610106, China
3
Sichuan Kelun Pharmaceutical Co. Ltd., Chengdu 610071, China
Correspondence should be addressed to Shu Yuan; roundtree318@hotmail.com
Received 9 April 2015; Revised 9 July 2015; Accepted 14 July 2015
Copyright 2016 Zhong-Wei Zhang et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Reactive oxygen species (ROS) play a crucial role in the inflammatory response and cytokine outbreak, such as during virus
infections, diabetes, cancer, cardiovascular diseases, and neurodegenerative diseases. Therefore, antioxidant is an important
medicine to ROS-related diseases. For example, ascorbic acid (vitamin C, VC) was suggested as the candidate antioxidant to treat
multiple diseases. However, long-term use of high-dose VC causes many side effects. In this review, we compare and analyze
all kinds of mitochondrion-permeable antioxidants, including edaravone, idebenone, -Lipoic acid, carotenoids, vitamin E, and
coenzyme Q10, and mitochondria-targeted antioxidants MitoQ and SkQ and propose astaxanthin (a special carotenoid) to be
the best antioxidant for ROS-burst-mediated acute diseases, like avian influenza infection and ischemia-reperfusion. Nevertheless,
astaxanthins are so unstable that most of them are inactivated after oral administration. Therefore, astaxanthin injection is suggested
hypothetically. The drawbacks of the antioxidants are also reviewed, which limit the use of antioxidants as coadjuvants in the
treatment of ROS-associated disorders.
1. Introduction mitochondrion-permeable antioxidants through analyzing

their therapeutic mechanisms, the application ranges, and
Endogenous reactive oxygen species (ROS) were produced side effects.
in cells over time, causing oxidative-damage to nucleic acids,
protein, lipids, and other cellular components. ROS are now
considered as signalling molecules to change the expression 2. ROS-Burst-Mediated Acute Diseases
of a large number of genes [1]. The relationship between
2.1. ROS Burst in Ischemia-Reperfusion. When blood supply
some diseases and oxidative-damage has been well studied.
or oxygen supply returns to the ischemic tissue, the reperfu-
A large number of reports showed that oxidative stress
is correlated with the pathogenesis of multiple age-related sion injury occurs. In this condition, restoration of blood flow
diseases, like cancer and neurodegenerative diseases, and and oxygen supply does not restore cellular normal functions
several other common diseases such as ischemia-reperfusion but induces inflammation and oxidative-damage [3, 12].
injury, stroke, hypertension, heart failure, atherosclerosis, Reperfusion of ischemic tissues is usually accompanied
diabetes, rheumatic diseases, and Alzheimer disease [210]. with microvascular damage, which increases capillary and
Therefore, a lot of antioxidants have been adopted to pre- arteriole permeability and leads to fluid filtration and dif-
vent and alleviate disease-accompanying oxidative-damage. fusion. These damaged endothelial cells generate more ROS
However, some human clinical data of antioxidant thera- but less nitric oxide after reperfusion, and the disequilibrium
peutics indicated negative or ambiguous results or insignif- induces subsequently inflammatory responses [3, 12, 13].
icant benefits. Even some antioxidants showed apparent side At the same time, leukocytes, circulated with the newly
effects [10, 11]. In this review, we compare representative returning blood, release interleukins, free radicals, and other
inflammatory factors, which damage the tissue further [3, induces the survival pathways, while hydrogen peroxide
12, 13]. The reintroduced oxygen damages nucleic acids, triggers the cell-death pathways [24]. Thus antioxidants may
enzymes, and the plasma membrane. Oxidative-damaged block both TNF and Bax-mediated apoptosis pathways [2].
cellular membrane may release more ROS in turn. Then
ROS may also trigger redox signalling indirectly and the 2.3. Vitamin C May Be Potentially Used as the Candidate
subsequent cell death or apoptosis. Leukocytes may also bind Antioxidant to Treat Avian Influenza Infections. In view of
to the small capillary endothelium, causing more ischemia the advantages such as relatively effective, nontoxic, and easy
[3, 12, 13]. to be absorbed, ascorbic acid (vitamin C, VC) was suggested
as the candidate antioxidant for avian influenza infections
2.2. ROS Burst in Avian Influenza Infections. The infection of [2]. VC scavenges free radicals through a nonenzymatic
avian influenza virus (AIV) results in multiple complications process. In the 19th century, VC was used to cure cold
to the patient, causing multiorgan failures and may be (influenza infection), encephalitis, hepatitis, and some other
associated with the excessive immune responses, which may viral diseases for over a hundred years [2527].
be the main reason for its high pathogenicity and mortality An investigation indicated that 50% of H5N1-infected
[2, 10]. The AIV infection induces a cytokine storm, including patients in Vietnam did not die. Ely [28] found that the
chemokines, interferon-inducible protein IP-10, interferon survivals may take large amounts of VC from their foods,
, and interleukin-6 (IL-6) and cell death presumably [14 which may alleviate the inflammatory responses.
17]. Investigations suggested that healthy young people with Influenza patients need 4.4 g or higher levels of VC to
stronger immune system may become a main target of AIV control the virus or alleviate the symptom [2528]. However,
attacks [2]. the common oral dosage of VC tablets is 100300 mg a day,
In our previous review of the drugs to avian influenza much lower than the influenza treatment requires. Oral
infection, a large drug combination (including antioxi- intakes of VC that exceed 1 g may cause side effects, like
dants, protectant of mitochondrial membrane permeability, vomiting, stomach cramps, diarrhea, and nausea [2528].
immunomodulators, protease inhibitors, and antiviral drugs) Therefore the VC injection should be used for AIV infections.
is proposed, which mainly focuses on cytokine control and Nevertheless, high doses are still required. Additionally, long-
may greatly reduce the mortality rate hypothetically [2]. For term use of high level of VC (>2-3 g a day) may result in
the drug combination, antioxidant is the most important scurvy after VC administration is stopped [2527]. These
medicine suggested, because of the fact that ROS play a drawbacks should be considered before the clinical therapies.
crucial role in the inflammatory response and cytokine
outbreak [18]. Neutrophil aggregation and oxidative-damage 2.4. Other ROS-Related Airway Disorders, Chronic Obstruc-
to alveolar epithelial membrane result in acute respiratory tive Pulmonary Disease, for Example. Chronic obstructive
distress syndrome (ARDS) finally. The activated neutrophils pulmonary disease (COPD) is a major and rapidly increas-
induce a ROS burst (more than ten times explosion). ing health problem associated with a chronic inflamma-
There are many similarities between pulmonary tory response, predominantly in small airways and lung
ischemia-reperfusion and AIV infection-induced ARDS. parenchyma. Oxidative stress induced by reactive oxygen
Both of the oxidative injuries include the following: (i) lipid species and nitrogen species plays a central role in the
peroxidation and oxidative-damage to cytomembrane and pathophysiology of COPD [29]. At the subcellular level,
the organelle-membrane; (ii) enzyme activity inhibition; mitochondrial dysfunction (accompanied with a decreased
(iii) lysosomal protease releasing; and (iv) chemoattractant mitochondrial membrane potential) in patients with COPD
generation and aggregation of more neutrophils [19]. Thereby is associated with excessive mitochondrial ROS levels, which
free radicals form a self-amplification feedback loop [18]. contribute to enhanced inflammation and cell hyperpro-
H1N1 infection also inhibits patients catalase expression, liferation. Thus, targeting mitochondrial ROS represents a
therefore causing the hydrogen peroxide accumulation [20], promising therapeutic approach in patients with COPD, such
while H5N1 triggers extracellular calcium influx, which as the mitochondria-targeted antioxidant MitoQ (see later
induces apoptosis [21]. discussion of MitoQ) [30].
Major inflammatory response is the mitochondrial dys-
function. AIV infection or ischemia-reperfusion induces 3. Mitochondrion-Permeable Antioxidants
calcium overload and mitochondrial permeability transi-
tion (mPT). Apoptosis indicator cytochrome c is released The most pivotal aspects of antioxidant therapies are the
from mitochondria. Cyclosporine A (CsA) can prevent this site concentration effects. Antioxidant efficiency is fully
mitochondrial permeability transition and the subsequent dependent on the locus concentration, since, as many other
apoptosis [22]. CsA-treated cells are protected from ischemia- pharmaceutical compounds, antioxidants also have their
reperfusion injury, but not from tumour necrosis factor pharmacological windows. Therefore, these scavenging/
(TNF) or Bax (Bcl-2 associated X protein) induced cell quenching compounds should concentrate in the target-
death [22]. Both ROS-mediated apoptotic pathway and NF- tissue (or subcellular site) in order to efficiently remove
B-mediated survival pathway are activated by the TNF. exceeding ROS without eliminating essential redox signalling
ROS accumulation facilitates the cell-death pathway [23]. The molecules, such as nitric oxide, hydrogen peroxide, S-Ni-
ratio of proapoptotic protein Bax to antiapoptotic protein trosoglutathione (GSNO), and nitro/nitrosyl-lipid peroxides
Bcl-2 is also regulated by the ROS level. Superoxide anion [24, 31]. It is well known that cellular redox status defines
Table 1: Licensed antioxidants for alleviating disease-related oxidative-damage. Their evidenced clinical uses, drawbacks, and possible side
effects are summarized.
Drugs name Clinical uses Drawbacks Possible side effects
Edaravone Limited testing and
Ischemic stroke Nephrotoxicity [95]
sometimes ineffective
Gastrointestinal complaints,
Idebenone Limited testing and
Alzheimer disease neurotoxicity, and cardiotoxicity
[95]
-Lipoic acid Diabetic neuropathy and eye-related Limited testing and Headache, tingling, skin rash, or
disorders sometimes ineffective muscle cramps [95]
Damage to skeletal muscle
integrity (high-dose) [44],
Carotenoids Inflammation, cancer, and
Sometimes ineffective canthaxanthin retinopathy [102],
cardiovascular diseases
and lung cancer in heavy smokers
[103]
Vitamin E Inflammation, cancer, and Hemorrhage and vitamin K
Sometimes ineffective
cardiovascular diseases deficiency (high-dose) [45]
Limited testing,
insoluble in water,
Coenzyme Q10 Heart failure, migraine, hypertension, Largely gastrointestinal complaints
therefore in low
and neurodegenerative diseases (very high-dose) [50]
bioavailability, and
Alzheimers disease, Parkinsons
disease, hypertension, diabetes, heart No side effect observed (even after
MitoQ Sometimes ineffective in
attack, sepsis, alcohol-induced a long-term oral administration)
human bodies
steatohepatitis, and cocaine [56]
cardiotoxicity
SkQ Age-related diseases Limited testing No side effect observed [59]
Atherosclerosis, coronary heart disease
and ischemic brain damage,
Astaxanthin Insoluble in water and
age-related macular degeneration, No side effect observed [6064]
acute pain, inflammation, cancer, and
cardiovascular diseases
the fate of one cell. Depending on the redox status, eukaryotic Therefore, they could traverse across the cell membrane and
cells could proliferate, keep it in steady state (G0 phase), or the mitochondrial membrane and accumulate in mitochon-
enter into cell death, either by apoptosis (moderate oxidative dria. On the contrary, most water-soluble antioxidants are
condition; intrinsic mitochondrial pathway) or by necrosis distributed in the cytosol (Figure 1).
(high oxidative insults) [24, 31]. More interestingly, the redox
status sensibility varies obviously upon the cell type that 3.1. Representative Mitochondrion-Permeable Antioxidants.
hepatic cells are more plastic than neurons [32]. Therefore, the Edaravone (3-methyl-l-phenyl-pyrazoline-5-one) has been
biggest challenge researchers have nowadays on prescribing approved in Japan since 2001. Edaravone can reduce
antioxidant therapies is how to reach the proper antioxidant ischemic-stroke-induced neuronal damage [33]. However,
concentration in situ for a precise redox modulation against there are also studies that do not approve the effects. Even
a ROS-mediated pathology. some cases of nephrotoxicity were reported for edaravone
As discussed above, ROS-burst-mediated mitochondrial [34].
dysfunction and mitochondrial-derived apoptosis play a cru- Idebenone (2,3-dimethoxy-5-methyl-6-(10-hydroxyde-
cial role in the inflammatory response during avian influenza cyl)-1,4-benzoquinonenoben) is a short chain benzoquinone,
infection or ischemia-reperfusion. Thus for these ROS-burst- structurally similar to coenzyme Q10. Idebenone functions as
mediated acute diseases, mitochondrion-permeable antiox- an antioxidant and electron carrier [35]. Although idebenone
idants should be much more effective than water-soluble has some effects on Alzheimers diseases [35, 36], the solid
antioxidants (like VC). Edaravone, idebenone, -Lipoic acid, clinical evidences are still missing. Therefore, its clinical
carotenoids (especially astaxanthin), vitamin E, coenzyme application is limited [37]. The most common side effects are
Q10, and mitochondria-targeted antioxidants MitoQ and gastrointestinal complaints and some level of neurotoxicity
SkQ are summarized as follows (Table 1 and Figure 1). Inter- or cardiotoxicity [38].
estingly, most of them contain a six-membered carbon-ring -Lipoic acid (LA) is a unique lipid and water-soluble
with a long alkyl side chain and multiple hydroxyl groups antioxidant. It is a naturally occurring dithiol compound
and aldehyde groups (Figure 1). All of them are liposoluble. and essential for mitochondrial bioenergetic process [39].
N O OH
N
Idebenone
O O
O
O
Edaravone
O
Coenzyme Q10
O O CH3
-Lipoic acid
H3 C
H3 C
HO O H
S S
O CH3 610
P+
O
MitoQ
O
HO
Vitamin E
O
-Carotene
OH
O
O
HO Astaxanthin
Figure 1: Chemical structures of representative mitochondrion-permeable antioxidants (edaravone, idebenone, -Lipoic acid, coenzyme
Q10, MitoQ, vitamin E, -carotene, and astaxanthin).
LA and its reduced-form dihydrolipoic acid are impor- However, for Parkinsons disease trials, MitoQ did not
tant mitochondrion-permeable antioxidants. LA has been show a benefit, maybe because of the irreversible neuronal
approved for diabetic neuropathy treatment [39, 40]. damage in patients brain cells [58]. Therefore, more studies
Carotenoids, consisting of over 600 lipid-soluble plant of MitoQ in humans are much needed. Moreover, only
pigments and a few water-soluble carotenoids (such as successful phase II assessments of oral MitoQ tablets were
crocin), are present in many fruits and vegetables. The com- reported. It is not a FDA-approved drug so far.
mon carotenoids include -carotene, -carotene, lycopene, SkQ (10-(6 -plastoquinonyl)decyltriphenyl-phosphoni-
-cryptoxanthin, lutein, and zeaxanthin [41]. Among them, um) also is organic molecules composed of a large number
-carotene, the vitamin A precursor, has been most well of organic cations attached with a plastoquinone. SkQ trav-
studied. They neutralize free radicals effectively [42]. How- erses across the cellular membranes and accumulates in
ever, there are inconsistent conclusions about the role of - mitochondria. The level of a penetrating cation in mito-
carotene in cardiovascular diseases (CVD) prevention [43]. chondria can be more than 1000-fold higher than its
Moreover, a study indicated that high intake of carotenoids extracellular level [59]. Therefore, it is another mitochondria-
resulted in a faster skeletal muscle breakdown (skeletal targeted antioxidant.
muscle integrity reducing) [44]. Astaxanthin is a peculiar Several studies indicated that SkQ protects cells from age-
carotenoid, which will be discussed in detail later. related diseases efficiently, including cataract, retinopathy,
Among the vitamin E family, -tocopherol is the most glaucoma, balding, canities, osteoporosis, hypothermia, and
predominant form. The hepatic -tocopherol transfer protein torpor [59]. However, its safety and the clinical usefulness
binds and carries -tocopherol to all bodys cells [45]. Most need further investigations. Like MitoQ, SkQ is also not a
-tocopherol is associated with lipoproteins, scavenging FDA-approved drug so far.
LCOO results and inhibiting low-density lipoprotein (LDL)
oxidation. Thus, -tocopherol is thought to have a role in
3.2. Astaxanthin Is a Promising Antioxidant. Better than
atherosclerosis prevention. The uptake process of oxidized
above antioxidants, here we introduce another one, astaxan-
LDL by the macrophage scavenger receptor and the foam cell
thin, to be a candidate drug for AIV infection cure and some
formation are blocked by the -tocopherol treatment [46].
other diseases (Table 1). Astaxanthin, a dietary carotenoid,
However, some reports did not support the protective role of
is present in algae, shrimp, lobster, crab, salmon, and some
vitamin E in prostate cancer [47, 48].
other organisms [6064]. Its antioxidant activity is far
Coenzyme Q10 (CoQ10), with its oxidized-form ubiquin-
exceeding the existing antioxidants. The ROS-scavenging
one and reduced-form ubiquinol, is an endogenous lipid,
capacity is 6000 times that of VC, 800 times that of coenzyme
which participates in the mitochondrial electron transport
Q10, 550 times that of VE, 200 times that of polyphenols, 150
in the respiratory chain [49]. CoQ10 has been used to
times that of anthocyanins, and 75 times that of -Lipoic acid
treat a variety of diseases, such as cardiovascular diseases
[65]. Most importantly, no apparent side effects or negative
[50], migraine [51], hypertension [52], and neurodegenerative
results have been reported for astaxanthin [6062]. In leuko-
diseases [53]. Although CoQ10 is considered a safe drug,
cyte cells, half of the total astaxanthin is distributed in the
further large-scale studies are still needed to show its clinical
mitochondria. Astaxanthin is also distributed in microsomes
usefulness.
and nuclei [66]. Therefore, it is a mitochondrion-permeable
MitoQ was designed in the late 1990s as a mitochondria-
antioxidant.
targeted antioxidant by Kelso et al. [54]. Both MitoQ and
coenzyme Q10 belong to the ubiquinone components. The Natural astaxanthin plays an important role in preventing
ubiquinone structure of MitoQ can be activated in the atherosclerosis. Low-density lipoprotein (LDL) oxidation is
mitochondrion (by the mitochondrial respiratory complex the main reason of atherosclerosis. Astaxanthin treatment
II) to form the ubiquinol antioxidant. Thus, MitoQ increases increased high-density lipoprotein (HDL) significantly and
the mitochondrial antioxidant capacity in situ and thereby reduced LDL effectively, while -carotene or canthaxanthin
decreases mitochondrial oxidative-damage [54]. has no such effect. The main reason may be that only
MitoQ is a lipophilic molecule bearing a cation moiety, astaxanthin can reduce apolipoprotein oxidation, therefore
which makes it pass directly through the mitochondrial being important for preventing arteriosclerosis, cardiovascu-
membrane, because of the fact that the component is pos- lar diseases, and ischemic brain damage [67, 68].
itively charged (a hydrophobic structure) [55]. Therefore, Astaxanthin also maintains the eyes and central nervous
MitoQ is an effective mitochondria-targeted antioxidant. system healthy. Retina contains high levels of unsaturated
The ability of MitoQ and the mitochondrial oxidative- fatty acids and oxygen supply. The singlet oxygen is generated
damage after the treatments (oral or intraperitoneal admin- in the retina upon high-energy light illumination. However,
istration) have been studied in the mouse model. The for mammals, carotenoids in diet are enough to maintain eye
following diseases have been studied: Alzheimers disease, health and can quench these free radicals [69]. Recent study
hypertension, type I diabetes, heart attack, sepsis, fatty liver indicated that astaxanthin can pass through the blood-brain
disease, alcohol-induced steatohepatitis, doxorubicin, and barrier and prevent retina cell oxidation [70]. Astaxanthin
cocaine cardiotoxicity [56, 57]. These findings are consistent also has a good effect on preventing and treating macular
with the conclusion that mitochondrial oxidative-damage degeneration [70].
is the potential therapeutic target in multiple diseases and Astaxanthin is an anti-inflammatory and pain reliever,
pathologies. blocking different biochemical factors that cause ouch and
pain [71]. More specifically, astaxanthin inhibits cyclooxy- On the other hand, ROS accumulation does not always
genase 2 (COX2) enzyme activities, which are related with correlate with disease onsets positively. Watson [96] postu-
many diseases, such as osteoarthritis, rheumatoid arthritis, lates that diabetes (especially the type 2 diabetes), dementias,
dysmenorrhea, and acute pain [72]. Astaxanthin and Cele- cardiovascular disease, and some cancers may develop, when
brex (another COX2 inhibitor) work cooperatively for some oxidative redox potential in the endoplasmic reticulum is too
diseases, which therefore were suggested to be taken both low to form normal disulphide bonds [9799]. Maintaining
together to alleviate oxidative-damage [72]. a certain level of ROS may be necessary for correct protein
Astaxanthin affects not only the COX2 signalling pathway folding with disulphide bonds, which may be associated
but also multiple cytokines, like nitric oxide, interleukin 1- with type 2 diabetes and Alzheimers disease or some other
, prostaglandin E2, C-Reactive Protein (CRP), NF-B, and diseases [96, 100, 101]. Thus, the antioxidants may produce
TNF [72]. A study also showed that astaxanthin is a useful negative or ineffective impacts on some diseases.
antioxidant to treat insulin resistance by protecting cells from Interactions of carotenoids (such as canthaxanthin) with
TNF and palmitate-induced oxidative-damage [73]. Recent the lipid membranes and the aggregation of this pigment
study suggested that astaxanthin also inhibits apoptosis in may be the factors enhancing canthaxanthin toxicity towards
alveolar epithelial cells via mitochondrial ROS signalling the macula vascular system, which leads to the further
pathways, also indicating its mitochondrial location [74]. development of canthaxanthin retinopathy [102]. And high
Astaxanthin also activates T-cell and inhibits autoim- and long-term beta-carotene supplementation may increase
mune reactions [75]. The risk of many different types of lung tumor rates in heavy smokers [103].
cancer can be significantly reduced by dietary intake of as-
taxanthin along with other carotenoids [7678]. In the mouse 5. Hypothesis of Astaxanthin Injection
breast cancer model, astaxanthin treatment caused higher
levels of apoptotic cancer cells and protective interferons, Side effects of antioxidants (antioxidant-induced stress) only
inhibiting tumor growth [79]. (i) Astaxanthin prevents cancer present when antioxidants overwhelm the bodys free radicals
initiation by alleviating DNA oxidative-damage [80, 81]. [11]. Thus, antioxidants should be used carefully for chronic
(ii) Astaxanthin promotes early check and elimination of diseases, such as diabetes and Alzheimers disease, when
cells undergoing malignant transformation by activating cellular ROS levels are not particularly high (no ROS bursts
immune surveillance [82]. (iii) Astaxanthin prevents cancer occur). However, for ROS-burst-mediated acute diseases,
cell growth in cells by boosting immune detection [83, such as avian influenza infection and ischemia-reperfusion,
84]. (iv) Astaxanthin inhibits rapid tumor cell growth by antioxidants should be used as early as possible to avoid
blocking tumor cell reproductive cycle and inducing tumor or retard excessive immune responses. Mitochondrion-
cell apoptosis [8587]. (v) Astaxanthin prevents tumor cell permeable or mitochondria-targeted antioxidants are pre-
spreading by decreasing tumor cells tissue-melting proteins ferred.
[84]. Astaxanthin is a good candidate drug for these acute
diseases. However, so far, astaxanthin is not a clinical drug
McNulty et al. [88] studied membrane structures of
but merely a health care product. Most studies showed that its
carotenoids and the relationship to their biological activities.
treatment effects are not as good as people expect, contrasting
They found that the vertical orientation of astaxanthin in
to its extraordinary high antioxidant activities [61]. One of
membranes may be crucial for its high efficiency on removing
the reasons may be that astaxanthin is usually applied by oral
aggressive free radicals from membranes, especially in the
administration (such as astaxanthin soft capsule). All nour-
presence of water-soluble antioxidants, such as glutathione
ishments and oral drugs are digested in the gastrointestinal
and/or ascorbic acid [88].
tract and then absorbed into gastric veins and intestinal veins
and transported to the liver through the portal vein. Then,
4. Side Effects of Antioxidants after the livers process, they are transported throughout the
body via heart and arteries. Astaxanthin is easy to be oxidized
Most of the so-called antioxidant compounds also develop that most of them are inactivated during the digestion,
prooxidant properties under specific conditions, such as absorption, and transportation. After avian influenza virus
ascorbic acid (>1 mM) that induces Fe(III) reduction to Fe(II) infection, for instance, severe oxidative-damage occurs at the
[89]. Epigallocatechin 3-gallate (EGCG) produces hydrogen lungs, where neither oral VC tablets nor oral astaxanthin
peroxide and hydroxyl radicals in the presence of Fe(III) [90]. capsules could reach effectively. For the same reason, active
Clinical trials of some antioxidants in humans showed (reduced) astaxanthin could not reach atherosclerosis sites,
negative or ambiguous results or insignificant benefits [10, retina, or brain arteries ideally too. Therefore the injection
11, 9195]. The reasons may be as follows. (i) Oxidative- approach of astaxanthin may be adopted to these patients.
damage is neither the primary cause nor the only cause It is well known that vitamin E injection (a mixture of
of the disease. (ii) Patients do not benefit from the same oil for injection and VE) is better absorbed by the body
antioxidant treatment equally. (iii) Some antioxidants by oral since it goes directly into the blood stream [60, 62]. And
administration are of lower efficiencies. (iv) Some antioxidant recent studies indicated that VC injections have strong
molecules have toxic effects that mask their ROS-scavenging anticancer effects, especially when intravenous glutathione or
activities. (v) Certain antioxidants are not effective in well- vitamin K3 was applied synergistically [104, 105]. A similar
nourished populations [11, 95]. astaxanthin injection could be easily developed. For AIV
infections, astaxanthin by injection can be quickly absorbed Authors Contribution

and go directly into the pulmonary alveoli, where inflam-
matory reactions occur, through the bodys blood circulation Zhong-Wei Zhang and Xiao-Chao Xu contribute equally to
system. this work.
It is well known that water- and lipid-soluble antioxidants
act in synergism to efficiently remove aggressive radicals Acknowledgments
from hydrophobic compartments and, thereby, inhibit lipid
peroxidation, which is extremely harmful to most organelles This work was supported by the National Natural Science
[106]. The collaborative mechanism involving -tocopherol Foundation of China (31300207), the Funds from Sichuan
through tocopheryl formation and ascorbic acid has been Province Ministry of Education (13ZB0296 and 014z1700),
studied since the middle of the 90s [106]. In other words, by and the Preeminent Youth Fund of Sichuan Province
combining lipid-soluble antioxidants (such as astaxanthin) (2015JQO045).
with water-soluble ones (such as ascorbic acid) in lower
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Oxidative Medicine and Cellular Longevity

Modulation of Oxidative Stress:

Modulation of Oxidative Stress:

Guest Editors: Liudmila Korkina, Tomris Ozben,

1,4-Dihydropyridine Derivatives: Dihydronicotinamide AnaloguesModel Compounds Targeting

Redox Control of Multidrug Resistance and Its Possible Modulation by Antioxidants

Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli

Modulation of Hypercholesterolemia-Induced Oxidative/Nitrative Stress in the Heart

Protective Effects of D-Penicillamine on Catecholamine-Induced Myocardial Injury

The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

Mitochondrion-Permeable Antioxidants to Treat ROS-Burst-Mediated Acute Diseases

Liudmila Korkina,1 Tomris Ozben,2 and Luciano Saso3

Correspondence should be addressed to Luciano Saso; luciano.saso@uniroma1.it

Received 11 January 2016; Accepted 12 January 2016

lycopene might have protective or cytotoxic effects in pho-

Chiara De Luca,1 Elena V. Mikhalchik,2 Maxim V. Suprun,2

Correspondence should be addressed to Liudmila G. Korkina; korkina@cibi-nanolab.com

Received 5 August 2015; Revised 6 December 2015; Accepted 24 December 2015

Academic Editor: Tiziana Persichini

1. Introduction dietary substances with potential antiageing efficacy is their

Number (%) of participants

Parameter Pretreatment period Before treatment After treatment

< 0.0001 versus before treatment.

Nitrites/nitrates (NO2 + NO3 , plasma, M) MDA (plasma, M)

Hydroxyproline (plasma, g/mL) Coenzyme Q10 (CoQ10H2 + CoQ10 , plasma, mg/L)

3.0 p < 0.00125

oxidation, thus maintaining inflammation characteristic for References

[71] A. Bindoli and M. P. Rigobello, Principles in redox signaling:

Received 14 August 2015; Accepted 7 October 2015

Academic Editor: Luciano Saso

1. Introduction in cancer cells in which, depending on various factors, OS

C2 H5 OOC COOC2 H5 H3 COOC COOCH3 H3 COOC COOCH2 CH(CH3 )2

H3 C N CH3 H3 C N CH3 H3 C N CH3

H3 C N CH3 H3 C N CH3 H3 C N CH2 OCH2 CH2 NH2

H3 C N CH3 H3 C N CH3 H3 C N CH3

H3 COOC COOCH(CH3 )2 H3 COOC COOC2 H5 C2 H5 OOC COOC2 H5

H3 C N CH3 H3 C N CH3 H3 C N CH3

NO2 NO2 NO2

NO2 NO2 NO2

H3 COOC COOCH2CH2 N N CHPh2 H3 COOC COO H3 COOC COOC(CH3)3

H3 C N SCH3 H3 C N SCH3 H3 C N SCH3 H3 C N SCH3

Scheme 1: Reactions of 1,4-dihydropyridines leading to the formation of pyridine derivatives.

Systems of LDL oxidation

in vitro [112], but also have beneficial effects on repairing 5-Acetyl(carbamoyl)-6-methylsulfanyl-1,4-DHP-carbo-

COOH CONHCH(CH2 )2 COONa CONH(CH 2 )3 COONa

C2 H5 OOC COOC2 H5 C2 H5 OOC COOC2 H5 C2 H5 OOC COOC2 H5

H3 C N CH3 H3 C N CH3 H3 C N CH3

Figure 4: Derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA).

[61] Reaxys, Database. synthesis of chiral compounds, in Biosystems Engineering I:

Correspondence should be addressed to Liudmila Korkina; korkina@cibi-nanolab.com

Received 12 August 2015; Revised 14 November 2015; Accepted 18 November 2015

Academic Editor: Sidhartha D. Ray

1. Introduction cells [2]. Frustrating the great expectations raised, ABC

Prx: Peroxiredoxins cytochrome P450 super-family in human extrahepatic tissues,

keratinocytes by interfering with activation of transcription

Valerio Chiurchi,1,2 Antonio Orlacchio,3,4 and Mauro Maccarrone1,2

Correspondence should be addressed to Valerio Chiurchiu; v.chiurchiu@hsantalucia.it

Received 12 August 2015; Accepted 18 October 2015

Academic Editor: Liudmila Korkina

Micol Falabella,1 Elena Forte,1 Maria Chiara Magnifico,1 Paolo Santini,1

Correspondence should be addressed to Paolo Sarti; paolo.sarti@uniroma1.it

Received 10 August 2015; Revised 19 October 2015; Accepted 25 October 2015

Academic Editor: Liudmila Korkina

1. Introduction clinically relevant phenotype in most of the patients is RGCs